curcumin-targeting pericellular serine protease matriptase...

Research Article

Curcumin-Targeting Pericellular Serine Protease MatriptaseRole in Suppression of Prostate Cancer Cell Invasion, TumorGrowth, and Metastasis

Tai-Shan Cheng1, Wen-Chi Chen5, Ya-Yun Lin1, Chin-Hsien Tsai3, Chia-I Liao1, Hsin-Yi Shyu1,4,Chun-Jung Ko1, Sheue-Fen Tzeng3, Chun-Yin Huang5, Pan-Chyr Yang2, Pei-Wen Hsiao3, andMing-Shyue Lee1

AbstractCurcumin has been shown to possess potent chemopreventive and antitumor effects on prostate cancer.

However, themolecularmechanism involved in curcumin’s ability to suppress prostate cancer cell invasion,

tumor growth, and metastasis is not yet well understood. In this study, we have shown that curcumin can

suppress epidermal growth factor (EGF)- stimulated and heregulin-stimulated PC-3 cell invasion, as well as

androgen-induced LNCaP cell invasion. Curcumin treatment significantly resulted in reduced matrix

metalloproteinase 9 activity and downregulation of cellular matriptase, a membrane-anchored serine

protease with oncogenic roles in tumor formation and invasion. Our data further show that curcumin is

able to inhibit the induction effects of androgens and EGF onmatriptase activation, as well as to reduce the

activated levels of matriptase after its overexpression, thus suggesting that curcumin may interrupt diverse

signal pathways to block the protease. Furthermore, the reduction of activated matriptase in cells by

curcumin was also partly due to curcumin’s effect on promoting the shedding of matriptase into an

extracellular environment, but not via altering matriptase gene expression. In addition, curcumin signif-

icantly suppressed the invasive ability of prostate cancer cells induced by matriptase overexpression. In

xenograft model, curcumin not only inhibits prostate cancer tumor growth and metastasis but also

downregulates matriptase activity in vivo. Overall, the data indicate that curcumin exhibits a suppressive

effect on prostate cancer cell invasion, tumor growth, and metastasis, at least in part via downregulating

matriptase function. Cancer Prev Res; 6(5); 495–505. �2013 AACR.

IntroductionProstate cancer is currently the second leading cause of

cancer-related death in men in the western world (1).Localized prostate cancer is highly curable by surgery orchemotherapy to remove or destroy the cancerous lesions.Yet, there currently exist no efficacious therapies foradvanced prostate cancer with hormone refractory or met-astatic phenotypes (2). Therefore, it is a critical issue todiscover a chemical or compound valuable for developingnew therapies that inhibit the progression or invasive abil-ities of prostate cancer.

It has been shown that the expression levels ofmatriptase,a membrane-anchored serine protease, are increased inprostate cancer and correlated with tumor grades and poorprognosis (3). Several agents including androgens, epider-mal growth factor (EGF), sphingosine-1-phosphate, andsuramin exhibit stimulatory effects on the matriptase acti-vation inmammalian epithelial, prostate, and breast cancercells (4–6). In matriptase transgenic mice, matriptase over-expression promoted tumorigenecity and carcinogen-induced tumor formation (7). Matriptase is also involvedin ErbB-2-induced prostate cancer cell invasion (6). Thissuggests that dysregulationofmatriptase exhibits oncogeniceffects and can also promote the progression of humancancer including prostate cancer. Inhibition of matriptasemaybe anopportunity to reduceprostate tumor growth andprogression (8). Thus, matriptasemay present a new poten-tial target for prostate cancer therapies.

Curcumin has emerged as a compound with multiplebiologic properties for health maintenance and cancerprevention. The antiinflammatory and antioxidant activi-ties of curcumin have been proposed via inhibiting NF-kB,COX-2, iNOS, and cytokine production (9, 10). In rodents,curcumin can prevent carcinogenesis induced by variouscarcinogens (11). A more recent phase II clinical study has

Authors' Affiliations: 1Department of Biochemistry and Molecular Biol-ogy,College ofMedicine; 2College ofMedicine,National TaiwanUniversity;3Agricultural Biotechnology Research Center, Academia Sinica; 4Investi-gation Bureau, Ministry of Justice, Taipei; and 5Graduate Institute ofIntegrated Medicine, China Medical University, Taichung, Taiwan

Corresponding Author: Ming-Shyue Lee, Department of Biochemistryand Molecular Biology, College of Medicine, National Taiwan University,R817, 8F, No. 1, Section 1, Jen-Ai Rd., Taipei, Taiwan. Phone: 011-886-2-2395-7966; Fax: 011-886-2-2391-5295; E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-12-0293-T

�2013 American Association for Cancer Research.

CancerPreventionResearch

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pointed out that allotting low concentrations of curcuminin patients have a similar biologic impact onNF-kB,COX-2,and phospho-STAT-3 in peripheral blood mononuclearcells, as those observed in the in vitro studies with 5 to 50mmol/L of curcumin treatments (12, 13). In hepatocellularcancer cells, curcumin can inhibit cancer cell invasion andmatrix metalloproteinase 9 (MMP-9) secretion (14). Inprostate cancer cells, curcumin can downregulate androgenreceptors (10, 15), decrease cell proliferation (16), andinhibit DU145 cell invasion via decreasingMMP-2/-9 activ-ity (17). However, the more detailed molecular mechan-isms how curcumin inhibits prostate cancer cell invasion,tumor growth, and metastasis have not been yet clearlyelucidated. In this study, we show that curcumin can sig-nificantly suppress prostate cancer cell invasion, tumorgrowth, and metastasis. Curcumin not only inhibitsMMP-9 activity, but also decreases the cellular levels ofmatriptase. Furthermore, the invasive ability of prostatecancer cells induced by matriptase overexpression was alsosignificantly suppressed by curcumin. In summary, the dataindicate that curcumin exhibits a suppressive effect onprostate cancer cell invasion, tumor growth, andmetastasis,at least in part by downregulating matriptase function.Thus, the data suggest that curcumin may exhibit a thera-peutic potential for invasive prostate cancer.

Materials and MethodsMaterials

DMEM, FBS, and RPMI1640 media were obtained fromHyclone. Protein Assay kits were from Bio-Rad. Anti-V5 Aband Lipofectamine�TM2000 reagent were purchased fromInvitrogen. Curcumin (Cat.# C7727, purity 94% curcumi-noid, 80% curcumin) and all other reagentswere purchasedfrom Sigma-Aldrich, unless otherwise noted. M24, M69,and M19 antibodies were gifts from Dr. Chen-Yong Lin atthe Georgetown University, DC.

Cell culturePC-3, CWR22Rv1, andWPMY-1 cells were obtained from

American Type Culture Collection (ATCC). PNT-2 cellswere purchased from Sigma-Aldrich. PC-3 and WPMY-1cells were maintained in Dulbecco’s modified Eagle’s medi-um (DMEM) containing 10% fetal bovine serum (FBS) and2 mmol/L glutamine in a 5% CO2 and incubated at 37�C.DU145, C-33, and C-81 LNCaP cells were gifts from Dr.Ming-Fong Lin at the University of Nebraska Medical Cen-ter,NE.CWR22Rv1,C-33,C-81 LNCaP,DU145, andPNT-2cells were maintained in 5% FBS, 2 mmol/L glutamineRPMI1640 medium. No authentication was done by theauthors for all cell lines used.

Cell growth and cytotoxicity assaysCellswere seeded at a density of 1.5�104or 3�105 cells/

cm2 for cell growth and cytotoxicity assays, respectively. Forgrowth assay, 1 day after seeding, cell amounts were dailyanalyzed by MTT assays according to the manufacturer’sprotocol, and other cells were accordingly refreshed withmedia. For cell cytotoxicity assay, 1 day after seeding, the

cells were treated with indicated concentrations of curcu-min for 16 hours and the viable cell numbers were countedby a hemacytometer with a trypan blue exclusion method.

Cell invasion and migration assaysTranswell assays were conducted according to the previ-

ously described procedures (18). In brief, transwells werecoated with or without 20 mg of matrigel (BD biosciences)for cell invasion or migration assay. Serum-starved cellswere then seeded at a density of 3 � 105 cells/cm2 in theupper chambers of transwells with serum-freemedium. Thelower chambers were filled with the medium containing10% FBS or growth factors as chemoattractants. After 16-hour incubation, cells were fixed and stained with 1%GIEMSA dye. The penetrating cells were photographed(100�) and counted using a light microscope. All experi-ments were conducted in triplicate.

RNA extraction and quantitative real-time PCRBoth assays were conducted according to the previously

described procedures (19). Briefly, 16-hour, curcumin-trea-ted cells were used for total RNA extraction using Trizolreagent (Invitrogen), and RNA was reversely transcribedusing SuperScript One-step RT-PCR System (Invitrogen)according to the manufacturer’s instructions. Quantitativereal-time PCR (Q-PCR) was conducted using the StepOneReal-time PCR system (Applied Biosystems). The primersfor the experiments were listed as follows: Matriptase:forward, 50-CACCTCAGTGGTGGCTTTCC-30 and reverse,50-GCGTGCAGGCCAAAGCT-30; GAPDH: forward, 50-AAAGGATCCACTGGCG TCTTCACCACC-30 and reverse,50-GAATTCGTCATGGATGACCTTGGCCAG-30. HAI-1 pri-mers were obtained from ABI (Cat.#Hs00173678). All Q-PCR reactions were carried out 3 times.

Gelatin zymographyFor gelatin zymography, cells were treated with different

concentrations of curcumin for 16 hours. The supernatantsof the conditioned media were collected and concentratedusing Amicon Ultra-4 centrifuge filter devices (Millipore) at3,000 r.p.m. at 4�C for 30 minutes. Without heating orreducing, samples underwent 0.1% (w/v) gelatin SDS-PAGE. After electrophoresis, the gel was incubated in arenaturation buffer (50 mmol/L Tris-HCl, pH7.5, 10mmol/L NaCl, 2.5% v/v Triton X-100) at 37�C for 1.5hours, and following with a developing buffer (50mmol/L Tris-HCl, pH7.5, 5 mmol/L CaCl2) at 37�C for18 hours with gentle agitation. Gels were then stained andclear bands were shown on a blue background. The bandswere detected by a luminescent image analyzer (LAS-4000;Fujifilm).

Western blot analysisForWestern blotting byM24,M69, andM19mAbs, equal

amounts of cell lysates weremixedwith protein loading dyein a nonreducing and nonboiling condition (20). For theother samples, equal amounts of cell lysates were mixedwith regular protein loading dye and boiled for 10minutes.

Cheng et al.

Cancer Prev Res; 6(5) May 2013 Cancer Prevention Research496




Samples were separated by SDS-PAGE and transferred tonitrocellular membranes (Whatman). The membraneswere blocked with 5% skim milk in Tris-buffered salineand tween 20 (TBST) at room temperature for 1 hour andthen incubated with primary antibodies at 4�C overnight,followed by secondary antibody incubation. The proteinimageswere visualizedusing anEnhancedLuminol ReagentPlus (Perkin Elmer) and detected by a luminescent imageanalyzer (LAS-4000; Fujifilm).

Tumor xenografts and bioluminescence analysisAll procedures for animal experimental protocols were

approved by Institutional Animal Care and Use Committee(IACUC) of Academia Sinica andNTU. For xenograft study,6-week-oldmale nudemice were inoculated subcutaneous-ly into the dorsal flank with 2 � 106 luciferase-expressedPC3 (PC3-Luc) cells. After 2 weeks, mice were randomlyassigned into 2 groups (6 mice/group): 1 group receiving100mg/kg of curcumin and the other receiving vehicle(corn oil) by daily intraperitoneal injection. The tumorvolume and body weight of each mouse was monitoredweekly. After 3-week treatment, mice were sacrificed andindividual tumors were taken, weighted, and snap frozen inliquid nitrogen for Western blot analysis. For ex vivo bio-luminescence images of lymph nodes, the images wereacquired with Xenogen IVIS50 Imaging System, and mea-surements of bioluminescent signals were conducted withLiving Image 2.50 software. D-Luciferin of 150 mg/kg wasinjected into themice 10minutes before imaging.Micewerehumanely sacrificed, and both brachial lymph nodes weretaken and imaged for 10 seconds.

Statistical analysisAmean� SE was calculated from 3 repeated groups in all

experiments. A statistical significance between groups wasdetermined by Student t test. A P-value below 0.05 wasconsidered as a significant difference between the 2 groups.

ResultsCurcumin inhibition of prostate cancer cell migrationand invasionTo examine the effects of curcumin on the growth of PC-3

cells, and ensure that the cell growth kept in the log phaseafter 5-day culture, cells were seeded at a density of 1.5�104

cells/cm2 and treated with 5, 25, and 50 mmol/L of curcu-min. With MTT assays, Fig. 1A shows that curcumin ablyinhibits PC-3 cell growth in a time- and dose-dependentmanner.We then tested the cytotoxicity of curcumin on PC-3 cells in a cell density of 3� 105 cells/cm2, because this celldensity would be used in following transwell assays. Usingtrypan blue exclusion assays, the results displayed that therewas no significant cytotoxicity on PC-3 cells after the cur-cumin treatment (Fig. 1B). That 25 or 50 mmol/L of curcu-min dramatically decreased PC3 cell growth without anysignificant effect on cell cytotoxicitymaybe partly due to thedifference of cell-seeding density and/or the inhibitoryeffect of curcumin on dehydrogenases (21). To furtherinvestigate the effect of curcumin on prostate cancer cell

migration and invasion, we conducted transwell assaysand found that the migration and invasion of PC-3 cellswere significantly decreased by approximately 50% after5 mmol/L-curcumin treatment and suppressed up to 80%upon 25 and 50 mmol/L curcumin treatments (Fig. 1C andD). Taken together, these data indicate that curcumin cansignificantly suppress PC-3 cell migration and invasion.

Since recent studies have shown that oncogenic signalingof EGFR/ErbB-2 participates in metastatic prostate cancer(22), the effect of curcumin on ErbB ligands (EGF or here-gulin)-induced prostate cancer cell invasion was furtheranalyzed. As shown in Fig. 1E, EGF or heregulin was ableto increase PC-3 cell invasion and this induced cancer cellinvasion was ably antagonized by curcumin. Since andro-gen signaling has been another important factor for prostatecancer progression, to further explore the effect of curcuminon androgen-induced prostate cancer cell invasion, wetreated androgen-sensitive LNCaP cells with DHT in thepresence or absence of curcumin. The result (Fig. 1F) hasshown that DHT significantly enhanced the invasion ofLNCaP cells and curcumin suppressed DHT-inducedprostate cancer cell invasion. Thus, the data indicate thatcurcumin can block ErbB ligands (EGF and heregulin)- andDHT-induced prostate cancer cell invasion.

Identification of matriptase as a curcumin-targetingserine protease in prostate cancer cells

It has been shown that both MMPs and serine proteasesplay important roles in cancer cell invasion (23, 24). Tofurther explore the involvement of MMPs or serine pro-teases in curcumin-inhibited prostate cancer cell invasion,we analyzed the effects of a broadMMP inhibitor GM6001,a serine protease inhibitor 4-(2-Aminoethyl) benzenesul-fonyl fluoride hydrochloride (AEBSF), or curcuminonPC-3cell invasion. As shown in Fig. 2A, curcumin, GM6001, andAEBSF suppressed the invasion of PC-3 cells approximatelyby 76%, 38%, and 70%, respectively.With the combinationtreatment of GM6001 and curcumin, curcumin furtherreduced the invasion of PC-3 cells approximately by 35%in comparison with GM6001 alone. Moreover, curcuminonly had a marginal effect to further reduce the invasion ofAEBSF-treated cells, and the degree of curcumin to inhibitprostate cancer cell invasion was quite similar to the com-bined effect of GM6001 and AEBSF on this event. The datasuggest that curcumin-inhibited prostate cancer cell inva-sion is mainly via suppressing both MMPs and serineproteases. Indeed, the activity of secretedMMP-9 in prostatecancer cells was significantly suppressed by curcumin, witha less effect on MMP-2 (Fig. 2B).

Since the expression of matriptase has been shown to becorrelated with the prostate cancer progression (3) and alsothat matriptase is involved in ErbB-2-induced prostatecancer cell invasion (6), we then proposed that matriptasewas a curcumin-targeting serine protease in prostate cancercells, by examining the effect of curcumin on matriptaseusing Western blotting with anti-total matriptase (M24),anti-activated matriptase (M69) and anti-HAI-1 (M19)mAbs. As shown in Fig. 2C, curcumin proficiently decreased

Curcumin-Suppressing Matriptase, Prostate Cancer Cell Invasion, and Metastasis

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the total levels ofmatriptase including latentmatriptase (70kDa) and activated matriptase (a 120 kDa complex ofactivated matriptase and its cognate inhibitor HAI-1) in adose-dependent manner. The decrease in matriptase/HAI-1complexes by curcumin was further validated by the West-ern blotting with M69 Ab. In addition, the protein levels ofHAI-1 or in a complex with activated matriptase were alsoreduced by curcumin. To further assess the effect of curcu-min on the transcription levels of matriptase andHAI-1, weconducted Q-PCR and showed that curcumin had no sig-nificant effect on the expression of both genes (Fig. 2D).Thus, the data indicate that the reduction of matriptase andHAI-1 protein levels by curcumin is not via transcriptionregulation, but may utilize a posttranslational mechanismfor decreasing cellular matriptase.

Since epithelial–mesenchymal transitions (EMT) havebeen shown to be implicated in the prostate cancer pro-gression (25), we then investigated if curcumin functionedas an antogonist for EMT to reduce prostate cancer cellinvasion, by using the immunoblotting analyses of severalEMT markers including E-cadherin, b-catenin, vimentin,

and snail. As shown in Fig. 2E, 25 mmol/L curcumin hadno significant effect on those biomarkers in PC-3 cells.

Next, we examined the time-kinetic effect of curcumin onmatriptase in PC-3 cells. The data showed that the levels ofactivated matriptase dramatically decreased at 30 minutesafter curcumin treatment, and up to 16 hours, whereas thelevel of latent matriptase began to increase after 8-hourtreatment, lasting to 24 hours (Fig. 2F). Moreover, todetermine whether this decrease in matriptase by curcuminwas a ubiquitous phenomenon in prostate cancer andprostate cells, we analyzed the effect of curcumin onmatrip-tase in various human prostate cancer cells, prostaticstromal myofibroblast WPMY-1 cells, and immortalizedprostatic epithelial PNT-2 cells. The result (Fig. 2G) hasshown that curcumin also suppress the activated levels ofmatriptase in androgen-sensitive C-33 LNCaP cells andandrogen-independent prostate cancer cells includingC-81 LNCaP, DU145, and CWR22Rv1 cells. Moreover,matriptase protein in WPMY-1 cells and the activated levelof matriptase in PNT-2 cells were negligibly detectable.Apparently, curcumin can reduce the level of latent

Figure 1. Effect of curcumin onprostate cancer (PCa) cellmigration and invasion. A, analysisof curcumin effect on PC-3 cellgrowth using MTT methods. B,examination of curcumin effect onthe cytotoxicity of PC3 cells with atrypan blue exclusion method.Curcumin effects on PC3 cellmigration (C) and invasion (D) wereanalyzed using cell migration orinvasion assays. Examination ofcurcumin effects on EGF-inducedand HRG-induced PC3 cellinvasion (E), and on androgen-induced LNCaP cell invasion (F).Each assay was conducted intriplicate and statisticallycalculated (mean � S.E.;��, P < 0.001).

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matriptase in PNT-2 cells (Fig. 2G). Thus, the data indicatethat curcumin exhibits an inhibitory role in matriptase inprostate cancer cells, with a less effect on prostatic epithelialPNT-2 cells.In addition, we examined the effects of 2 curcumin

analogues, dimethoxycurcumin (DMC) and EF24, onmatriptase in PC3 cells and found that both analogues canalso reduce the activated levels of matriptase (Fig. 2H).Taken together, these data suggest that curcumin-decreasedprostate cancer cell invasion is at least partly due to decreas-ing bothMMP-9 activity and cellularmatriptase, rather thanaltering the EMT process.

Curcumin-reduced matriptase function in PC-3 cellsNext, we examined the pretreatment effect of curcumin

on the FBS-induced matriptase activation in prostatecancer cells. The data have shown that the pretreatment

of curcumin dramatically depressed the stimulatoryeffects of FBS on matriptase activation (Fig 3A). To furtherevaluate the duration of curcumin on reducing cellularmatriptase in prostate cancer cells, the effect of 1-houracute curcumin treatment on matriptase was examined.The data showed that curcumin could decrease the acti-vated matriptase up to 8 hours after the acute treatment(Fig. 3B). Four hours after the acute treatment, thedecreased levels of latent matriptase began to rebound.We then analyzed the effect of the acute curcumin treat-ment on prostate cancer cell invasion and found that theacute treatments of 25 and 50 mmol/L curcumin sup-pressed PC3 cell invasion by approximately 50% andapproximately 64% (Fig. 3C). This result indicates thatan acute exposure of curcumin remains effective in reduc-ing both cellular matriptase and prostate cancer cellinvasion.

Figure 2. Identification ofmatriptase as a curcumin-targeting serine protease in prostate cancer cells. A, effects of curcumin,GM6001, andAEBSFonPC-3 cellinvasionwere analyzedandstatistically calculated (mean�S.E.; ��,P<0.001). B, curcumin's effect onMMPactivity usinggelatin zymographywasanalyzed intriplicate and statistically calculated (mean�S.E.; ��,P < 0.001). C, analysis of curcumin's effects onmatriptase andHAI-1 in PC3 cells using immunoblottingassays with M24, M69, and M19 mAbs. D, effects of curcumin on the gene expression of matriptase and HAI-1 in PC-3 cells were analyzed byQ-PCR. E, analysis of curcumin effects on the expression of E-cadherin, b-catenin, vimentin, and snail in PC3 cells by immunoblot assays. F, time-kineticeffect of curcumin on matriptase in PC-3 cells. Cells were treated with 25 mmol/L curcumin for indicated times for Western blot analysis with M24 andM69 Abs. G, effect of curcumin on matriptase in different prostate cancer and epithelial cells. LNCaP (LN, C-33, and C-81), DU145, CWR22Rv1 (22Rv1), andprostate epithelial PNT-2 cells were treated with or without curcumin for 16 hours and used for the immunoblot analysis. H, effects of 10 mmol/Ldimethoxycurcumin (DMC) and 5 mmol/L EF24 on matriptase in PC-3 cells were analyzed by immunoblot assay. b-Actin was used as control.






Inhibitory effects of curcumin on DHT- and EGF-induced matriptase activation

Since androgens can proficiently induce matriptaseactivation in prostate cancer cells (26), we then examinedthe effect of curcumin on androgen-induced matriptaseactivation in LNCaP cells. As shown in Fig. 3D, curcuminhad an inhibitory effect on DHT-induced matriptaseactivation in a dose-responsive manner. Moreover, weexamined if curcumin also exhibited an antagonizedeffect on EGF-induced matriptase activation in prostatecancer cells. As shown in Fig. 3E, EGF could dramaticallyinduce matriptase activation in LNCaP cells, and curcu-min ably reduced EGF-induced matriptase activation.Likewise, we further analyzed the acute effect of curcuminon EGF-induced matriptase activation. After the treat-ment with EGF for 2 hours and followed by curcumintreatment for 30 minutes, the results showed that curcu-min could quickly decrease the stimulatory effect of EGFon matriptase in LNCaP cells (Fig. 3F). These resultsdisplay that curcumin can potently inhibit the stimula-tory effects of androgens and EGF on matriptase inprostate cancer cells.

Curcumin promotion of matriptase shedding andsuppression ofmatriptase-inducedprostate cancer cellinvasion

The shedding of matriptase–HAI-1 complexes to extra-cellular environments is thought of as a mechanism toremove matriptase from cells (26). Since Fig. 2 shows thatcurcumin can reduce the cellular content of matriptasebut has no effect on the gene expression, we accordinglyexamined whether curcumin could promote the sheddingof matriptase, leading to decreasing cellular matriptase inprostate cancer cells. The effect of curcumin on matriptaseshedding was examined by using Western blotting toanalyze the shed matriptase in the conditioned media.The data revealed that curcumin promoted the sheddingof the matriptase–HAI-1 complex in a dose-dependentmanner, with less effect on the latent form of matriptase(Fig. 4A). We then further analyzed whether the shedmatriptase in the conditioned media retains its functionfor cancer cell invasion. Following the curcumin treat-ment, the conditioned media were collected, washed outof curcumin and used to treat PC-3 cells. Through cellinvasion assays, the data showed that the shed matriptase

Figure 3. Curcumin reducedmatriptase activation in prostate cancer cells. A, analysis of curcumin pretreatment on FBS-inducedmatriptase activation in PC-3cells. Cells were pretreated with the indicated concentrations of curcumin for 24 hours and recultured with the medium containing 10% FBS forindicated times. B, analysis of acute curcumin treatment onmatriptase in PC-3 cells. Cells received 1-hour curcumin treatment and regrew in a regular culturemedium for indicated times. Cell lysates were used for immunoblot analysis. C, examination of acute curcumin treatment on PC3 cell invasion. One-hour,curcumin-treated PC-3 cells were used for cell invasion assay with statistical calculation. ��, P < 0.001. D, analysis of curcumin effect on DHT-inducedmatriptase activation in C-33 LNCaP cells by immunoblot analysis. E, effects of curcumin on EGF-induced matriptase activation in C-33 LNCaP cells.Cells were treated with or without curcumin in the presence or absence of DHT (D) or EGF (E) for 16 hours, and used for immunoblot analysis. F, analysis ofacute curcumin treatment on EGF-induced matriptase activation in C-33 LNCaP cells. The cells were pretreated with EGF for 2 hours and then recieved anacute treatment of curcumin for 30 minutes. Cell lysates were used for immunoblot analysis.

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had no significant effect on prostate cancer cell invasion(Fig. 4B).Moreover, to recaptulate the increased levels of matrip-

tase in advanced prostate cancer, PC3 cells were transientlytransfected with V5-tagged matriptase plasmids. We thenanalyzed whether curcumin could also affect the activatedlevels of matriptase caused by overexpression. As shownin Fig. 4C, the overexpression of matriptase increased the

activated levels of matriptase, and curcumin effectivelyreduced the cellular levels of matriptase, at least in part bypromoting the shedding of the matriptase–HAI-1 complexinto the conditioned media. Thus, the data indicate thatcurcumin-inducedmatriptase sheddingmay play an impor-tant role in reducing the cellular level of activatedmatriptase.

Sincematriptase overexpression is observed in a variety ofhuman carcinomas including prostate cancer (3, 27), wefurther examined the role of matriptase in prostate cancercell invasion and the effect of curcumin on matriptase-induced cancer malignancy by establishing stable pools ofmatriptase-overexpressing CWR22Rv1 cells. The data dis-played that the total and activated levels of matriptaseincreased in matriptase-overexpressing CWR22Rv1 cells(Fig. 4D), which were concurrent with increased invasioncapabilities (Fig. 4E). We then examined the effect of cur-cumin and AEBSF on the invasion of matriptase-overex-pressing CWR22Rv1 cells. As shown in Fig. 4F, similar toAEBSF, curcumin significantly inhibited the matriptase-induced prostate cancer cell invasion, up to 80%, down tothe levels of curcumin-treated control cells. Thus, the datadenote that curcumin exhibits a strong inhibitory potentialfor prostate cancer cell invasion, especially that caused bymatriptase overexpression or dysregulation.

Curcumin-inhibited tumor growth and metastasis in aprostate cancer xenograft model

To further assess if curcumin can inhibit prostate tumorgrowth andmetastasis, we examined the effects of curcuminon the tumor growth of PC3-Luc cells using a xenograftmodel. As shown in Fig. 5A, there was a significant regres-sion of tumor volume in the group of mice receiving thecurcumin treatment. No animal body weight was signifi-cantly altered during the treatment, indicating that thecurcumin treatment had no toxicity in mice (Fig. 5B). Afterscarification, the tumormass wasmeasured and significant-ly reduced in the group of curcumin-treated mice (Fig. 5C).Moreover, we checked if there was anymetastatic nodule inthe lymphnodes nearby the xenografted tumors, and foundthat curcumin treatment significantly reduced the metastat-ic lesions at brachial lymph nodes in the xenografted mice,indicating that curcumin exhibited an inhibition effect onprostate cancer metastasis (Fig. 5D). We then further ana-lyzed the total and activated levels of matriptase in thexenografted tumors with or without curcumin treatment.As shown in Fig. 5E, the level of activated matriptase wasremarkably deceased in the xenografted tumors treatedwithcurcumin, compared with vehicle control. Taken together,the data indicate that curcumin can inhibit prostate cancertumor growth and metastasis, at least in part due to curcu-min’s effect on downregulating the activated matriptase intumors.

DiscussionRecent findings have recommended curcumin as a ben-

eficial chemotherapeutic and chemopreventive agent for

Figure 4. Curcumin promoted matriptase shedding and reducedmatriptase-induced prostate cancer cell invasion. A, analysis ofcurcumin effect on matriptase shedding in PC-3 cells. The conditionedmedia of 16-hour, curcumin-treated PC-3 cells were collected andconcentrated. Cell lysates and the conditioned media were analyzed byimmunoblot analysis. B, examination of shed matriptase on prostatecancer cell invasion. The conditioned media (CM) of 16-hour, 50 mmol/Lcurcumin-treated PC3 cells were collected, washed out of curcumin, andused for cell invasion assay. C, effectsof curcumin on the cellular and shedmatriptase in matriptase-overexpressing PC3 cells. PC-3 cells weretransiently transfected with V5-tagged matriptase plasmids (MTX),and their cell lysates and conditioned media were collected forimmunoblot analyses. The serum-free media (SFM) were used as acontrol. D, immunoblotting analysis of matriptase in V5-tagged MTX-overexpressedCWR22Rv1cells. Stable pools ofMTX-V5plasmids (MTX)-transfected CWR22Rv1 cells were selected by 400 mg/mL G418. Celllysates were prepared for immunoblot analyses. E, examination ofmatriptase role in prostate cancer cell invasion. CWR22Rv1 (C), vector(Vec)-transfected, and matriptase (MTX)-transfected CWR22Rv1 cellswere used for cell invasion assays with statistical calculation of mean �SE. F, effects of curcumin and AEBSF on matriptase-induced prostatecancer cell invasionwith statistical calculation ofmean�SE. ��,P < 0.001.






prostate cancer (28). However, its functions in prostatecancer cell invasion and metastasis are still lacking. In thisstudy, we found that curcumin exhibits an inhibitory effecton prostate cancer cell invasion, tumor growth, and metas-tasis, not only in terms of inhibiting MMP-9 activity, butalso on the downregulation of cellular matriptase, achievedthrough inhibiting the stimulatory effects of androgens orEGFR ligands on the protease and promoting the proteaseshedding.

For cancer cell invasion or metastasis, the EMT and thedegradation of ECM have been proposed as 2 importantprocesses. ECM degradation has been mainly attributed todysregulation of pericellular proteolysis. In curcumin-inhibited prostate cancer cell invasion, our results suggestthat some proteases from the families of MMPs and serineproteases are critically inhibited by curcumin, whereas theEMT is apparently not affected by this treatment. Moreover,our data indicate that in prostate cancer cells, MMP-9activity is significantly inhibited by curcuminwith less effecton MMP-2. Unexpectedly, we further observed that thecombination treatment of GM6001 and AEBSF could reachto the similar inhibitory effect of curcumin on prostatecancer cell invasion, implying that some serine protease(s)was curcumin-targeting and involved in promoting pro-state cancer cell invasion. In prostate cancer, ErbB-2 isoverexpressed in approximately 80% of metastasized pros-tate cancer lesions (29) and matriptase is involved in ErbB-2–induced prostate cancer cell invasion (6). These previousfindings inspired us to explore whether matriptase canpromote prostate cancer cell invasion and if curcumin caninhibit matriptase-induced prostate cancer cell invasion.Interestingly, our data revealed that matriptase overexpres-

sion can induce prostate cancer cell invasion, suggestingthat the dysregulation of matriptase is a factor in allowingcancer cells to obtain high invasion capabilities during theprostate cancer progression. Comparatively, our data fur-ther suggest that curcumin can inhibit matriptase-inducedprostate cancer cell invasion through reducing the cellularmatriptase. In addition, curcumin also can inhibit prostatetumor growth and metastasis, at least partly via downregu-lating the level of activated matriptase in prostate cancerxenografted tumors (Fig. 5). Thus, curcumin exhibits achemopreventive and therapeutic potential in matriptase-involved prostate cancer malignancy.

As shown in Fig. 6, matriptase function is mainly regu-lated by 3 steps: activation, inhibition, and ectodomainshedding. Matriptase is synthesized as a single-chain zymo-gen, through 2 sequential endoproteolytic cleavages foractivation: the first cleavage at G149 for matriptase matu-ration (30) and the second cleavage at Arg614 for activation(4). Matriptase overexpression, HAI-1 knockdown, S1P,EGF, Heregulin, or DHT can induce the protease activation(4–6, 26, 31). After activation, 2 mechanisms are proposedfor inhibiting matriptase activity: (i) its inhibitor HAI-1–mediated inhibition by forming 120-kDa complexes and(ii) the ectodomain shedding of the activated matriptase–HAI-1 complex from cells into the extracellular environ-ments with a molecular mass of 95 or 110 kDa (30, 32).Based on this model, we propose 2 possible mechanisms inwhich curcumin candownregulate cellularmatriptase. First,perhaps curcumin promotes the shedding process to reducethe cellular levels of matriptase, leading to decreased pros-tate cancer cell invasion. This suggests that curcumin’sability to decrease the cellular content of matriptase

Figure 5. Curcumin suppression oftumor growth and metastasis in aPC3 xenograft model. A, effect ofcurcumin on the tumor volumes ofPC3-Luc cell xenograft. The tumorvolumes in vehicle-treated (control;n ¼ 6) and curcumin-treated(100mg/kg/day; n ¼ 6) mice wereplotted and calculated as mean �S.E. ��,P<0.01. B, themousebodyweight after curcumin treatmentwas weekly monitored. C, after3-week treatment, tumor weightswere measured, plotted, andstatistically calculated as mean �SE. D, curcumin effect on tumormatastasis to brachial lymphnodes was examined bybioluminescent images as photonflux per second. E, whole cellhomogenates from xenograftedtumor tissues with vehicle andcurcumin treatment wereimmunoblotted. b-Actin wasused as loading control.

Cheng et al.





provides a way to reduce the protease’s oncogenic effect onprostate cancer. However, how curcumin enhances thematriptase shedding is still elusive and remains underfurther investigation. Second, curcumin can interrupt thestimulatory effects of EGF, heregulin, and androgens in theprocess of matriptase activation. This may be explained bycurcumin’s multiple inhibitory effects on several signalmolecules including androgen receptor, HER-2, EGFR,PI3K/Akt, etc. (9, 10, 33). Thus, curcumin may block theandrogen- and ErbB ligand-induced signal pathways formatriptase activation, leading to reducing prostate cancercell invasion. In conclusion, the data suggest that curcuminmay be a potent phytocomponent able to efficiently inhibitmatriptase-mediated prostate cancer malignancy.The inhibitory effects on the activated matriptase in

prostate cancer cells after an acute curcumin treatment onlylast up to 16 hours. This may be due to curcumin consump-tion by the cells or the instability of curcumin in the culturemedium, since curcumin has been shown to be unstable atneutral and basic pH and decomposed approximately by50% in the culture medium after 8-hour incubation (34).Therefore, 2 curcumin analogs (DMC and EF24) with anenhanced metabolic stability and a higher potency thancurcumin (35, 36) were used in this study, and had shownthat both analogs can also reduce the cellular levels ofactivatedmatriptase in prostate cancer cells. Thus, this studypoints out that enhancing the stability and potency ofcurcumin will improve the efficiency of curcumin in thechemoprevention or cancer therapy, especially in cancerswith matriptase dysfunction.Curcumin has been shown to downregulate urokinase-

type plasminogen activator (uPA) in prostate cancer cells

(37). Pro-uPA has been shown to be one of matriptasesubstrates (38, 39). Consequently, curcumin’s ability toinhibit the activities of uPA may be partly attributed to itsinhibitory effects on matriptase.

In conclusion, our data reveal that matriptase can beactivated by multiple factors, including its overexpression,growth factors or androgens, and ultimately lead to prostatecancer cell invasion. Curcumin can significantly decreasethe cellular levels of matriptase in prostate cancer cells, bypromoting protease shedding. Therefore, this study pro-vides a novel mechanism for establishing how curcumincan inhibit prostate cancer cell invasion, tumor growth andmetastasis by the downregulation of matriptase function.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: T.S. Cheng, Y.Y. Lin, C.I. Liao, M.S. LeeDevelopment of methodology: T.S. Cheng, P.W. HsiaoAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): T.S. Cheng, Y.Y. Lin, C.H. Tsai, C.I. Liao, S.F.Tzeng, P.W. Hsiao, M.S. LeeAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): T.S. Cheng, C.J. Ko, P.W. Hsiao, M.S. LeeWriting, review, and/or revision of themanuscript: T.S. Cheng, Y.Y. Lin,M.S. LeeAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases):W.C. Chen, Y.Y. Lin, H.Y. Shyu, C.J.Ko, P.C. Yang, M.S. LeeStudy supervision: W.C. Chen, Y.Y. Lin, P.C. Yang, M.S. Lee

AcknowledgmentsWe thank Dr. Chen-Yong Lin, Lombardi Cancer Center, Georgetown

University, for gifts of anti-matriptase and anti-HAI-1 antibodies, Dr. Ming-Fong Lin at the Department of Biochemistry and Molecular Biology, Uni-versity of Nebraska Medical Center, for gifts of prostate cancer cells, Dr. Jen-

Figure 6. Model of curcumin's effects onmatriptase in prostate cancer cells.Matriptase is synthesized as a single-chain polypeptide and its activation requires2 sequential endoproteolytic cleavages: the first cleavage at G149 for matriptase maturation (30) and the second cleavage at R614 for activation (4).Activematriptase is quickly inhibited byHAI-1with formation of 120 kDa complexes. Then, thematriptase–HAI-1 complex is shedwith amolecularmass of 95or 110 kDa into extracellular environments. The mechanism for curcumin to affect matriptase activity is proposed as follows: First, curcumin inducesmatriptase shedding, leading to decreasing cellular matriptase and prostate cancer cell invasion. Second, curcumin may inhibit matriptase activation bytargeting multiple signaling pathways, such as androgens, EGF, and heregulin signaling, even by its overexpression.






Kun Lin in our department for his help and comments for this study, andMs.Emily Lin for manuscript editing.

Grant SupportThis study was supported by Taiwan National Health Research Institutes

Grants NHRI-EX101-9909BC and NHRI-EX102-9909BC, Taiwan NationalScience Council Grants NSC 97-2320-B-002-052-MY3, NSC 100-2628-B-002-004-MY4, and NSC 101-2324-B-002-015, and the National Taiwan

University Cutting-Edge Steering Research Project 10R71602C4 to M.S. Lee;the Cooperative Research Program of NTUCM and CMUCM toM.S. Lee andW.C. Chen. Postdoctoral Fellowship from the Aim for the Top UniversityProgram, National Taiwan University and the Excellent Translational Med-icine Research Project (101C101-53) to T.S. Cheng.

Received July 6, 2012; revised February 20, 2013; accepted February 22,2013; published OnlineFirst March 6, 2013.

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2013;6:495-505. Published OnlineFirst March 6, 2013.Cancer Prev Res Tai-Shan Cheng, Wen-Chi Chen, Ya-Yun Lin, et al. MetastasisSuppression of Prostate Cancer Cell Invasion, Tumor Growth, and Curcumin-Targeting Pericellular Serine Protease Matriptase Role in

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