role of e-cadherin in antimigratory and antiinvasive efficacy of

Research Article

Role of E-cadherin in Antimigratory and AntiinvasiveEfficacy of Silibinin in Prostate Cancer Cells

Gagan Deep1,2, Subhash Chander Gangar1, Chapla Agarwal1,2, and Rajesh Agarwal1,2

AbstractThe epithelial-to-mesenchymal transition (EMT) in prostate cancer (PCA) cells is considered pre-

requisite for acquiring migratory/invasive phenotype, and subsequent metastasis. We hypothesized that

promoting the E-cadherin expression in PCA cells by using nontoxic phytochemicals, like silibinin,

would prevent EMT and consequently invasiveness. Our results showed that silibinin treatment

(5–90 mmol/L) significantly inhibits migratory and invasive potential of advance human PCA PC3,

PC3MM2, and C4–2B cells in in vitro assays. Importantly, the antimigratory/antiinvasive efficacy of

silibinin was not due to its cytotoxicity toward PCA cells. Molecular analyses showed that silibinin

increases E-cadherin level that was localized mainly at cellular membrane as evidenced by subcellular

fractional and confocal analyses in PC3 cells, which might be responsible for morphologically observed

shift toward epithelial character. Silibinin also decreased the levels of Slug, Snail, phospho-Akt(ser473),

nuclear b-catenin, phospho-Src(tyr419) and Hakai; together they play an important role in regulating

E-cadherin expression/function and EMT. Similar silibinin effects on E-cadherin, b-catenin, phospho-Src(tyr419), and Hakai levels were also observed in PC3MM2 and C4–2B PCA cells. Selective Src

inhibition by dasatinib also showed increased E-cadherin expression in PC3 cells suggesting a possible

involvement of Src inhibition in silibinin-caused increase in E-cadherin level. Additional studies in

PC3 cells with stable knock-down of E-cadherin expression revealed that antimigratory/antiinvasive

efficacy of silibinin is in-part dependent on E-cadherin expression. Together, our results showing

antimigratory/antiinvasive effects of silibinin and associated mechanisms suggest that silibinin should

be tested further in clinically relevant animal models toward exploiting its potential benefits against

metastatic PCA. Cancer Prev Res; 4(8); 1222–32. �2011 AACR.

Introduction

Prostate cancer (PCA) is the most commonly diagnosednoncutaneous malignancy, and is second leading cause ofcancer-related deaths in American men (1). Patients withlocalized PCA have a very high 5-year survival rate and arelatively lowmortality to incidence ratio as compared withother cancers. Patients with clinically detectable metastaticdisease, however, have median survival of 12–15 months,suggesting that cancer cell metastasis is the main cause ofhigh mortality among PCA patients (2–4). Currently, che-motherapy, radiotherapy, hormone therapy, biologicaltherapy, surgery, or a combination of these therapies areemployed to treat advance stage PCA patients; but in mostcases, these therapies provide only a marginal or no survi-

val benefit. Further, exorbitant costs as well as unacceptablelevel of toxicity associated with current therapeutic regimecall for newer measures to control PCA. One emergingapproach in cancer management is the use of nontoxic andbiologically effective dietary and nondietary agents. The listof these agents with efficacy against cancer metastasis isgrowing steadily and many of these agents have alreadyentered clinical phase (5–7).

Silibinin, a flavonoid from milk thistle seed extract, is awidely consumed dietary supplement for its efficacy againstliver disorders (8, 9). Silibinin has also shown strongefficacy both in vitro and in vivo against PCA cells, and iscurrently being evaluated in PCA patients (7, 10–14).Earlier, we reported the strong antimetastatic efficacyof silibinin in TRAMP (transgenic adenocarcinoma ofthe mouse prostate) model (12, 13); however, detailedmechanisms for its strong antimetastatic efficacy remainslargely unknown.

Metastasis is one of the hallmarks of cancer cells and isconsidered responsible for more than 90% of cancer-asso-ciated deaths (15). This is an extremely complex biologicalevent during which cancer cells acquire motility, invadelocally and enter into systemic blood circulation, survive incirculation, arrest in microvasculature and subsequently

Authors' Affiliations: 1Department of Pharmaceutical Sciences, School ofPharmacy, University of Colorado Denver and 2University of ColoradoCancer Center, Aurora, Colorado

Corresponding Author: Rajesh Agarwal, University of Colorado Denver,12850 E. Montview Blvd, C238, Aurora, CO 80045. Phone: 303-724-4055;Fax: 303-724-7266; E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-10-0370

�2011 American Association for Cancer Research.

CancerPreventionResearch

Cancer Prev Res; 4(8) August 20111222

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Published OnlineFirst May 5, 2011; DOI: 10.1158/1940-6207.CAPR-10-0370

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extravasate and grow at distant organs (2, 16, 17). Inmetastasis, acquisition of motility and invasiveness arethe major earlier events during which cancer cells shedmany of their epithelial characteristics, undergo drasticmodifications in their cytoskeleton and acquire highlymotile and invasive mesenchymal phenotype (18, 19).This phenomenon in cancer cells is known as epithelial-to-mesenchymal transition (EMT) and represents onemajor mechanism in cancer cell metastasis (18, 19).The molecular basis of EMT is very complex and involves

several interconnected pathways that downregulate theexpression of epithelial molecule E-cadherin, which is wellknown for regulating cell-to-cell contact, cell shape, andpolarity (18, 20). E-cadherin connects adjacent cellsthrough homophilic interactions and is also linked tothe cytoskeleton though multicatenin complex attachedto their cytoplasmic tails (21, 22). In this complex, b-cate-nin and p120 are directly associated with E-cadherin,whereas a-catenin is the link between b-catenin and actinmicrofilament network of the cytoskeleton (21, 22).Importantly, the aberrant or decreased expression of E-cadherin is considered as one of the biomarkers for poorprognosis in PCA (23, 24). Therefore, promoting E-cad-herin expression using nontoxic phytochemicals should beconsidered an ideal strategy toward preventing cancer cellsfrom acquiring motility and invasiveness.Lately, several transcriptional factors (Snail, Slug, Zeb1,

Twist, etc.) have also been identified which negativelyregulate E-cadherin expression and play important rolein EMT induction and maintenance of migratory andinvasive phenotype in cancer cells (18–20). A variety ofkinases such as MAPKs, PI3K, and Src are also known toregulate EMT and metastasis of cancer cells (18, 19). Aktis also reported to upregulate Snail and b-catenin ex-pression, thereby promoting EMT (18, 25). Src is a non-receptor tyrosine kinase whose overexpression andactivation has been associated with numerous types ofcancers including PCA (26, 27). Src is considered as anintegrator of several cellular signaling cascades in PCAcells, thereby it affects a wide range of biological phe-nomena including proliferation, migration, adhesion,and metastasis (26, 27). Due to its pleiotropic roles ingrowth and progression, various Src inhibitors are beingtested in clinic against PCA (26, 27). Src has also beenreported to phosphorylate E-cadherin that facilitates itsbinding with Hakai, a RING finger-type E3 ubiquitinligase, which leads to ubiquitination, endocytosis, andlysosomal-mediated degradation of E-cadherin (28, 29).Accordingly, in the present study, we assessed silibinineffect on migratory and invasive potentials of threehuman metastatic PCA cell lines namely PC3, PC3MM2,and C4–2B, and examined the role of E-cadherin andother EMT regulators in the biological efficacy of silibi-nin. Our findings showed a strong antimigratory andantiinvasive efficacy of silibinin against PCA cells, whichwas in-part through promoting E-cadherin expressionand decreasing the level of Slug, phosphorylated-Akt,nuclear b-catenin, phosphorylated-Src, and Hakai.

Materials and Methods

Cell lines and reagentsPC3 cells were from American Type Culture Collection

(ATCC). Highly metastatic PC3MM2 cell line was a kindgift (Dr. Isaiah J. Fidler, University of Texas M. D. AndersonCancer Center) and was originally selected from PC3 cells.C4–2B cells were derived from the bone metastasis ofLNCaP-variant cell line C4–2 and purchased from ViroMedLaboratories. All three cell lines were obtained during2008, and tested and authenticated by DNA profiling forpolymorphic short tandem repeat (STR) markers at Uni-versity of Colorado Cancer Center DNA Sequencing &Analysis Core most recently in August 2010. Antibodiesfor b-catenin (sc-7199, 1:1000 dilution), Vimentin(sc-7557, 1:250 dilution), Zeb1 (sc-25388, 1:200 dilution),Histone H1 (sc-10806, 1:500 dilution), and Hakai(sc-101912, 1:200 dilution), and E-cadherin shRNA plas-mid were from Santa Cruz Biotechnology. Antibodies forE-cadherin (#3195, 1:1000 dilution), phospho-Src(tyr419)(#2101, 1:1000 dilution), total Src (#2109, 1:1000 dilu-tion), phospho-Akt(Ser473) (#9271, 1:500 dilution), andtotal Akt (#4685, 1:1000 dilution), and antirabbit peroxi-dase-conjugated secondary antibody (#7074, 1:1000 dilu-tion) were obtained from Cell Signaling. Silibinin,mitomycin C, puromycin, and b-actin antibody (A2228,1:2000 dilution) were from Sigma–Aldrich. AmershamEnhanced Chemiluminescence (ECL) detection systemand antimouse horseradish peroxidase (HRP)-conjugatedsecondary antibody (NA931V, 1:1000 dilution) were fromGE Healthcare. Antibodies for Snail (ab17732, 1:500 dilu-tion) and Slug (ab27568, 1:500 dilution) were fromAbcam. GeneJuice transfection reagent was from Novagen.Dasatinib was purchased from Selleck Chemicals.RPMI1640 media and other cell culture materials werefrom Invitrogen Corporation. Antibody for a-tubulin(MS-581, 1:1000 dilution) was from Lab Vision Corpora-tion. All other reagents were obtained in their commerciallyavailable highest purity grade.

Cell culture and treatmentsPC3, PC3MM2, and C4–2B cells were cultured in

RPMI1640 medium supplemented with 10% heat inacti-vated fetal bovine serum and 100 U/ml penicillin G and100 mg/ml streptomycin sulfate at 37�C in a humidified 5%CO2 incubator. Cells were treated with different concen-trations of silibinin (5–90 mmol/L in medium), dissolvedoriginally in dimethyl sulfoxide (DMSO), for descried timeperiods. An equal amount of DMSO (vehicle) was presentin each treatment, including control; DMSO concentrationdid not exceed 0.1% (v/v) in any treatment.

Wound healing assayThe antimigratory efficacy of silibinin was examined

using the well-established wound healing assay (30, 31).Briefly, PC3 or PC3MM2 cells were grown to full con-fluence in six-well plates. In each case, cells were madequiescent by pretreating with 0.5 mmol/L mitomycin C for

Silibinin Inhibits PCA Migration/Invasion

www.aacrjournals.org Cancer Prev Res; 4(8) August 2011 1223




2 hours to ensure that wounds are filled due to cellmigration and not by cell proliferation (32, 33). Subse-quently, cells were wounded by pipette tips and washedtwice with media to remove detached cells, and photo-micrographs of initial wounds were taken using CanonPower Shot A640 digital camera (at 100� magnification).Thereafter, cells were treated with DMSO or 5–90 mmol/Lconcentrations of silibinin in RPMI1640 media (10% FBSand 0.5 mmol/L mitomycin C). Experiment was terminatedas soon as wound was completely filled in DMSO-treatedcontrols and photomicrographs of final wounds weretaken for each group. Initial and final wound sizes weremeasured using AxioVision Rel.4.7 software, and differ-ence between the two was used to determine migrationdistance using the formula: Initial wound size minus finalwound size divided by 2.

Invasion assayInvasion assay was conducted using matrigel coated

trans-well chambers from BD Biosciences as describedearlier (34). Briefly, in this assay, bottom chambers werefilled with RPMI media with 10% FBS and top chamberswere seeded with 1 � 105 cells (PC3, PC3MM2, or C4–2B)per well in RMPI media (with 0.5% FBS) along with DMSOor 5–90 mmol/L concentrations of silibinin. After 22 hoursof incubation under standard culture conditions, PCA cellson top surfaces of the membrane (noninvasive cells) werescraped with cotton swabs and cells on bottom sides ofmembrane (invasive cells) were fixed with cold methanol,stained with hematoxylin/eosin andmounted. Images weretaken using Cannon Power Shot A640 camera on Zeissinverted microscope and invasive cells were manuallycounted at 400� in 10 random fields on each membrane.

Migration assayMigration assay for C4–2B cells was carried out similar to

invasion assay described above, but in this assay the trans-well chambers (BD Biosciences) lacked matrigel layer (30).

Cell viability assayApproximately, 5� 104 cells (PC3, PC3MM2, or C4–2B)

were plated and treated with silibinin (5–90 mmol/L) inRPMI1640 media under normal serum condition. After22 hours of treatment, cells were collected and total cellnumber was determined by counting each samplein duplicate using a hemocytometer under an invertedmicroscope.

Western blottingTotal or nuclear/cytoplasmic cell lysates were prepared as

descried earlier (35, 36) and subcellular fractionations wereprepared as per vendor’s protocol (ThermoFisher Scienti-fic). For Western blotting, 40–70 mg of protein per samplewas denatured in 2� SDS–PAGE sample buffers and sub-jected to SDS-PAGE on 8 or 12% polyacrylamide tris-glycine gels followed by immunoblotting as describedearlier (36, 37). In each case, protein loading was mon-itored by stripping and reprobing the blots with b-actin or

a-tubulin antibody. The autoradiograms/bands werescanned and where mentioned, mean density ofbands was determined using Adobe Photoshop 6.0 (AdobeSystems).

Confocal imagingPC3 cells were grown on cover slips and treated with

silibinin (30–90 mmol/L). After 72 hours, cells were fixed in3.7% formaldehyde, permeabilized with 0.1% Triton X-100, blocked under 5% serum condition, and incubatedovernight with E-cadherin antibody. Next, cells were incu-bated with FITC-tagged secondary antibody along with 40,6 diamidino 2 phenylindole (DAPI) for 30 minutes.Images were captured at 1000� magnification on a Nikoninverted confocal microscope using 488/405 nm laserwavelengths to detect FITC (green) and DAPI (blue) emis-sions, respectively. Images were quantified for FITC-green color based upon following scoring criterion: 0þ(no color), 1þ (dim color), 2þ (moderate color), 3þ(bright color), 4þ (very bright color).

TransfectionPC3 cells were transfected at about 50% to 60% con-

fluency using GeneJuice as per vendors’ protocol. Briefly,0.5 mg of random shRNA plasmid or 0.5 mg of E-cadherinspecific shRNA plasmid was incubated with GeneJuice/serum free mediummixture for 15minutes and then addeddrop-wise to cells. Transfected cells were selected throughpuromycin-based selection, and PC3 cells with stableknock-down of E-cadherin (ShEC-PC3 cells) and respectivecontrol cells (Sh-PC3 cells) were identified.

Real-time reverse transcription-PCRTotal RNA was isolated using PerfectPure RNA cultured

cell kit from 5 Prime and E-cadherin mRNA level wasquantified by real-time reverse transcription (RT)–PCRfollowing the procedure detailed earlier (38). The primersused for E-cadherin were 50-AGTGTCCCCCGGTATCTTCC-30 and 50-CAGCCGCTTTCAGATTTTCAT-30. Quantity of E-cadherin mRNA in each sample was normalized to thecorresponding 18S rRNA level.

Statistical analysisStatistical analysis was carried out using SigmaStat 2.03

software (Jandel Scientific). Data was analyzed using one-way ANOVA (Tukey test) and statistically significant dif-ference was considered at P � 0.05.

Results

Silibinin exerts strong antimigratory and antiinvasiveefficacy against prostate cancer cells

Silibinin treatment (5–90 mmol/L) inhibited the migra-tory potential of PC3 and PC3MM2 cells by 30%–76%and 18%–80%, respectively, in a dose-dependent manner(Figs. 1A and 1B). In C4–2B cells, creation of woundirreversibly affected their migratory potential and woundwas not filled within experimental duration; therefore, we

Deep et al.

Cancer Prev Res; 4(8) August 2011 Cancer Prevention Research1224




studied silibinin effect on migratory potential of C4–2Bcells using migration chambers. In this assay, silibinintreatment inhibited (13%–87%) migratory potential ofC4–2 B cells in a dose-dependent manner (Fig. 1C).Next, we examined effects of silibinin on the invasive

potential of PC3, PC3MM2, and C4–2B cells in well-estab-lished invasion assay. This is an excellent in vitro model tostudy the potential of cancer cells to invade through extra-cellular matrix, which is the first barrier during cancer cellsinvasion. Results showed that silibinin inhibits invasivepotential of PC3, PC3MM2, and C4–2B cells by 11%–65%,20%–62% and 28%–55%, respectively, in a dose-depen-dent manner (Figs. 2A-2C). Next, we examined effect ofsilibinin treatment on the viability of PCA cells after 22hours of treatment. As shown in Fig. 2D, silibinin treatment(5–90 mmol/LM) decreased viability by 9%–34% in PC3,8%–43% in PC3MM2 and 8%–29% in C4–2B cells

(Fig. 2D). The cell viability data suggest that silibininhas antimigratory and antiinvasive efficacy at concentra-tions where it has minimal, if any, cytotoxicity toward PCAcells.

Silibinin targets signaling molecules regulatingepithelial-to-mesenchymal transition in PC3 cells

To understand possible mechanism/s underlying silibi-nin antimigratory and antiinvasive efficacy, we first exam-ined its effect on E-cadherin expression. As shown inFig. 3A, silibinin treatment (30–90 mmol/L) increasedthe protein expression of E-cadherin after 24, 48, and72 hours. Real-time RT-PCR assay showed that E-cadherinmRNA level was increased by approximately 1.8- foldafter 72 hours of silibinin treatment, whereas no changein E-cadherin mRNA level was observed after 24 and48 hours (data not shown).

Figure 1. Silibinin inhibitsmigratory potential of human PCAcells. A–B, effect of silibinintreatment on migratory potentialof PC3 and PC3MM2 cells wasanalyzed through wound healingassay. Representativephotomicrographs of initial andfinal wounds are shown at 100xmagnification. Cell migrationdistance data shown are mean�SEM of three samples. C, effectof silibinin treatment on themigratory potential of C4–2B cellswas evaluated using migrationchambers. Cell migration datashown is mean�SEM of foursamples. These results (A–C) weresimilar in 2–3 independentexperiments. (SB, silibinin;*, P � 0.001; #, P � 0.01;$, P � 0.05.)

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Next, we examined silibinin effect on other major reg-ulators and markers of EMT. As shown in Fig. 3A, silibinintreatment decreased the protein expression of Slug after24, 48, and 72 hours whereas a decrease in Snail expres-sion was observed only after 24 and 48 hours. Further,silibinin treatment decreased the Akt phosphorylation atser-473 site after 48 and 72 hours but only marginallyaffected total Akt level. Silibinin treatment did not affectthe levels of Zeb1 and mesenchymal marker proteinVimentin after 24, 48, and 72 hours in PC3 cells (datanot shown).

Effect of silibinin on the expression and subcellulardistribution of E-cadherin and b-catenin in PC3 cells

In epithelial cells, b-catenin is generally localized tobasolateral plasma membrane, and in association with E-cadherin plays a critical role in cell–cell adhesion (22).Loss of E-cadherin during EMT releases b-catenin from themembranous pool, making it available for nuclear signal-ing, which then promotes cancer-cell proliferation and

invasiveness (21, 39). Therefore, next we examined sili-binin effect on b-catenin level in nuclear and cytoplasmicfractions of PC3 cells. As shown in Fig. 3B, silibinintreatment significantly decreased b-catenin level innuclear fraction of PC3 cells after 24, 48, and 72 hourswithout affecting b-catenin level in cytoplasmic fraction.Next, we prepared cytoplasmic, membrane, and nuclearfractions to analyze silibinin effect on the localization aswell as expression levels of E-cadherin and b-catenin.Silibinin treatment resulted in a significant increase inE-cadherin expression in the membrane fraction of PC3cells (Fig. 3C), but caused a strong decrease in b-cateninlevel in nuclear fraction with only a slight decrease incytoplasmic fraction and no significant change in mem-brane fraction (Fig. 3C). Blots were stripped and reprobedwith Histone H1 to confirm purity and equal proteinloading in nuclear fractions. Blots were also reprobedwith Akt antibody as a loading control for cytoplasmicandmembrane fractions. We selected this protein becausesilibinin treatment for 24 hours did not significantly alter

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Figure 2. Effect of silibinin on theinvasiveness and viability ofhuman PCA cells. A–C, effect ofsilibinin treatment on invasivepotential of PCA cells wasexamined using invasionchambers. Cell invasion datashown are mean�SEM of foursamples. These results weresimilar in two independentexperiments. D, effect of silibinintreatment (22 hours) on cellviability was measured as detailedin "Materials and Methods". Eachbar is representative of mean�SEM of three samples for eachtreatment. (SB, silibinin;*, P � 0.001; #, P � 0.01;$, P � 0.05.)

Deep et al.





the Akt level in total cell lysates (Fig. 3A). Confocalanalysis also confirmed that silibinin (30–90 mmol/L)promotes E-cadherin expression especially at cellularmembrane in a dose-dependent manner in PC3 cells

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Next, we examined the effect of silibinin on E-cadherinand b-catenin levels in PC3MM2 and C4–2B cells. As

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Figure 3. Silibinin targets signaling molecules regulating EMT in PCA cells. A, PC3 cells were treated with DMSO or silibinin at 40% to 50% confluency.Cells were harvested 24, 48, and 72 hours later, lysates prepared and Western blotting was carried out for E-cadherin, Slug, Snail, phospho-Akt(ser473)and total Akt. B, PC3 cells were treated with DMSO or silibinin for indicated time. Cells were harvested, nuclear, and cytoplasmic fractions were prepared andWestern blotting was carried out for b-catenin. C, PC3 cells were treated with silibinin for 24 hours and cytoplasmic, membrane, and nuclear fractionswere separated and Western blotting was carried out for E-cadherin and b-catenin. Effect of silibinin treatment on E-cadherin was also examined throughconfocal microscopy and representative pictures are shown where FITC-green is for E-cadherin whereas DAPI-blue stains nuclei. FITC-green quantificationdata shown is mean�SEM of three samples. #, P � 0.01; $, P � 0.05 D, PC3MM2 and C4–2B cells were treated with DMSO or silibinin at 40%–50%confluency. Cells were harvested 24 hours later, lysates prepared and Western blotting was carried out for E-cadherin and b-catenin. (SB, silibinin; kDa,kilo Dalton.)






shown in Fig. 3D, silibinin treatment increased E-cadherinprotein expression in both PC3MM2 and C4–2B cells,whereas slightly decreased b-catenin level in total celllysates, supporting that silibinin effects on E-cadherinand b-catenin are not PCA cell line specific.

Silibinin decreases Hakai level and Srcphosphorylation in prostate cancer cells

As shown in Fig. 4A, silibinin treatment (30–90 mmol/L)decreased the Hakai level in PC3 cells after 24, 48, and72 hours. Similarly, silibinin treatment (30–90 mmol/L)for 24 hours decreased the Hakai level in both PC3MM2and C4–2B cells; even though decrease was more promi-nent in the latter cell line (Fig. 4B). Next, we examinedsilibinin effect on Src phosphorylation at tyrosine-419 sitethat is considered surrogate marker for Src activity (27).

Silibinin treatment (30–90 mmol/L) strongly decreasedphospho-Src(tyr419) after 24, 48, and 72 hours in PC3cells without affecting total Src level (Fig. 4A). In PC3MM2cells, decrease in phospho-Src(tyr419) was prominentonly at 60 and 90 mmol/L concentrations of silibinin,whereas in C4–2B cells silibinin was also effective at30 mmol/L concentration, without affecting total Src levelin any treatment (Fig. 4B).

To further dissect Src role in the regulation of E-cadherinexpression, we next used dasatinib, a well-known pharma-cologic inhibitor of Src. As shown in Fig. 4C, dasatinib (25–100 nmol/L) strongly decreased the level of phospho-Src(tyr419) after 24 hours of treatment. Importantly, inhibi-tion of Src phosphorylation by dasatinib was accompaniedwith increased E-cadherin level in PC3 cells (Fig. 4C).Cell morphologic analysis revealed that dasatinib and/or

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Figure 4. Silibinin decreasesHakai and Src phosphorylationlevel and Src inhibitor dasatinibincreases E-cadherin expressionin PCA cells. (A-B, PCA cells weretreated with DMSO or silibinin andafter desired time points, Westernblotting was carried out for Hakai,phospho-Src(tyr419), and total Src.C, PC3 cells were treated withdasatinib (25–100 nmol/L) andafter 24 hours of treatment celllysates were analyzed forphospho-Src(tyr419) andE-cadherin. D, PC3 cells weretreated with dasatinib (50 or 100nmol/L) and/or silibinin (90 mmol/L)for 24 hours and morphologicchanges were captured under alight microscope at 100�magnification. Arrows in thepictures highlight therepresentative small- and round-type cells with epithelialcharacteristics. Subsequently,total cells lysates were analyzedfor E-cadherin protein expression.Densitometry data presentedbelow the bands are "fold change"as compared with control (DMSOtreated) after normalization withrespective loading control(tubulin). (SB, silibinin; Das,dasatinib; kDa, kilo Dalton.)

Deep et al.





silibinin treatment decrease/s the number of elongated andfibroblast-like cells (a mesenchymal characteristic) whileincrease/s the number of small and round-type cells (anepithelial characteristic) compared with DMSO treatedcontrol PC3 cells (Fig. 4D). To dissect whether silibininincreases E-cadherin level by other pathways independentof Src, we inhibited Src phosphorylation by dasatinibtogether with silibinin treatment. Western blotting analysisof the lysates showed that when Src phosphorylation isselectively inhibited, silibinin treatment does not affect E-cadherin level, suggesting a key role of Src inhibition bysilibinin in the observed increase in E-cadherin level.

Antimigratory and antiinvasive efficacy of silibinin isregulated in-part by upregulation of E-cadherinexpression in PC3 cells

To further understand the role of silibinin-causedincrease in E-cadherin in its antimigratory and antiinva-sive potential, we generated PC3 cells with stable knock-down of E-cadherin expression (ShEC-PC3 cells) andrespective control cells (Sh-PC3 cells). E-cadherin knock-down was confirmed in ShEC-PC3 cells with or withoutsilibinin treatment through Western blotting (Fig. 5A).As shown in Fig. 5A, E-cadherin knock-down resultedin increased level of phospho-Src(tyr419), which was

Figure 5. Effect of silibinintreatment on migratory andinvasive potential of PC3 cells withstable knock-down of E-cadherinexpression. A, Sh-PC3 cells orShEC-PC3 cells were treated withDMSO or silibinin for 24 hours,lysates prepared and Westernblotting was carried out forE-cadherin, phosphorylated, andtotal Src. B, representativepictures comparing themorphology of Sh-PC3 andShEC-PC3 cells are shown at200�magnification. C—D, woundhealing and invasion assays wereconducted with Sh-PC3 andShEC-PC3 cells. The data shownare mean�SEM of three samples,and results were similar in twoindependent experiments.(SB, silibinin; kDa, kilo Dalton;*, P � 0.001; #, P � 0.01;$, P � 0.05.)

A

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inhibited by silibinin treatment. Morphologic analysisshowed that ShEC-PC3 cells were elongated, spindle-shaped, and more mesenchymal in appearance comparedwith Sh-PC3 cells (Fig. 5B).

Next, we compared the antimigratory efficacy of silibininin Sh-PC3 and ShEC-PC3 cells using wound-healing assay.As shown in Fig. 5C, E-cadherin knock-down significantlyincreased the migratory potential of ShEC-PC3 cells com-pared with respective control Sh-PC3 cells (P � 0.05). InSh-PC3 cells, silibinin treatment inhibited migratorypotential by 28% (P � 0.001), 39% (P � 0.001), 69%(P � 0.001), 75% (P � 0.001), and 89% (P � 0.001) at 5,10, 30, 60, and 90 mmol/L concentrations, respectively(Fig. 5C). In ShEC-PC3 cells, however, silibinin treatmentinhibited migratory potential by 19% (statistically non-significant), 26% (statistically nonsignificant), 48% (P �0.01), 55% (P � 0.001), and 76% (P � 0.001) at 5, 10, 30,60, and 90 mmol/L concentrations, respectively (Fig. 5C).These results showed that under E-cadherin knock-downconditions, silibinin antimigratory efficacy is partially com-promised.

Next, we compared the antiinvasive potential of silibininin Sh-PC3 and ShEC-PC3 cells using invasion assay. Asshown in Fig. 5D, E-cadherin knock-down significantlyincreased the invasive potential of ShEC-PC3 cells com-pared with respective control Sh-PC3 cells (P � 0.001). InSh-PC3 cells, silibinin treatment (90 mmol/L) inhibitedinvasive potential approximately by 66% (P � 0.01);whereas in ShEC-PC3 cells, silibinin (90 mmol/L) inhibitedinvasive potential approximately by 37% (P � 0.01)(Fig. 5D). These results showed that silibinin antiinvasiveefficacy is also partially compromised under E-cadherinknock-down condition.

Discussion

In majority of PCA cases, death occurs due to cancermetastasis to bones (40, 41). Therefore, there is urgent needto develop new and better strategies toward preventing ortreating metastatic stage of this disease. One approach inthe management of advance PCA could be the use ofnatural agents also known as "nutraceuticals", which arerelatively nontoxic, cost-effective, physiologically bioavail-able, and have multiple molecular targets in cancer cells(6, 42–44). Earlier, we reported in TRAMP mice thatnutraceutical agent silibinin targets EMT-related regulatorsas well as many proteases and prevents metastatic spread ofPCA cells to distant organs (12, 13). In cell culture studies,silibinin was reported to decrease migratory/invasivepotential and to inhibit Vimentin and increase cytokera-tin-18 expression in PCA cells (31, 45, 46). Despite thesefindings, the main molecular target/s responsible forsilibinin’s strong antimigratory and antiinvasive efficacyremained inconclusive. In that perspective, results frompresent study are highly significant as we report for the firsttime that (a) silibinin inhibits invasive and migratorypotential of three highly metastatic PCA cell lines at non-cytotoxic concentrations; (b) silibinin increases E-cadherin

expression at cellular membrane and inhibits nuclearb-catenin level; (c) silibinin decreases phospho-Src(tyr419), Hakai, Slug, Snail and phospho-Akt(ser473) levels;and (d) antiinvasive and antimigratory effects of silibininare in-part through promoting E-cadherin expression inPCA cells. These findings have great translational relevancebecause the concentrations of silibinin used in these studiesare within the range of free silibinin levels achieved in theplasma of PCA patients in our completed phase I clinicaltrial (14).

The present study has shown that silibinin significantlydecreases nuclear b-catenin level without affecting its levelon cellular membrane where it is known to bind with E-cadherin and promote cell-to-cell contact. Therefore, itseems plausible that silibinin treatment increases E-cad-herin level at cellular membrane as well as promotes E-cadherin and b-catenin association; thereby restore cell-to-cell contact which is detrimental to migratory and invasivepotential of PCA cells. Future studies are needed to under-stand silibinin’s effect on such molecular interactionsbetween E-cadherin and b-catenin.

Receptor and nonreceptor kinases, such as Met andSrc respectively, have been reported to promote thephosphorylation of intracellular tail of E-cadherin attyrosine sites, which is then recognized by ubiquitin-ligase protein Hakai mediating its internalization andubiquitin-dependent degradation (28, 29). Interestingly,overexpression of Hakai alone could enhance E-cadherinendocytosis, disrupts cell–cell adhesion, and could pro-mote cell motility (28). Besides directly phosphorylatingE-cadherin, activated Src could also phosphorylateb-catenin, reducing its association with E-cadherin andpromoting its nuclear localization, thereby, severely dis-rupting the adhesion complex (21, 47). In Fig. 6, we have

E-Cadherin

Phosphorylated E-cadherinSrctyr419

Srctyr419

β-Catenin

Phosphorylated β β-catenin

Increased nuclear translocationand transcriptional activity

Loss of cell–cell contactIncreased migratory potentialIncreased invasiveness

Hakai

Ubiquitination and endocytosis ofE-cadherin

E-Cadherin degradation

Increased by silibinin treatment

Inhibited by silibinin treatment

Figure 6. Proposed molecular targets in antimigratory and antiinvasiveefficacy of silibinin in PCA cells. Silibinin targets Src and Hakai mediatedE-cadherin degradation as well as nuclear translocation of b-catenin;thereby prevents loss of cell–cell contact and inhibits migratory andinvasive potential of PCA cells.

Deep et al.





depicted these molecular interactions involving Src,Hakai, b-catenin and E-cadherin, which result in loss ofcell–cell contact and enhanced migratory and invasivebehavior in PCA cells. In the present study, we found thatsilibinin decreases Src activity in terms of its tyrosinephosphorylation and Hakai expression in PCA cells;thereby silibinin could limit Src capability to phosphor-ylate E-cadherin or b-catenin and inhibit the resultantE-cadherin degradation and nuclear translocation ofb-catenin (Fig. 6). Overall, these molecular effects ofsilibinin’s might be responsible for observed enhancedcell–cell contact and decreased migratory and invasivepotential of PCA cells with silibinin treatment (Fig. 6).Results from present study showed that silibinin-

caused upregulation of E-cadherin in-part contributesto antimigratory and antiinvasive efficacy of silibinin,suggesting that other signaling molecules could also con-tribute toward silibinin’s biological efficacy againstmetastatic PCA cells. Our initial studies showed that E-cadherin knock-down results in increased Src phosphor-ylation that was significantly inhibited by silibinintreatment and might be responsible for the remnantbiological effects of silibinin in the absence of E-cadherin.Further studies are needed in future to understandwhether the effect of silibinin on Src activation is director through targeting the molecules that are upstream of

Src such as integrins and epidermal growth factor receptor(EGFR) which are also known to get activated in responseto E-cadherin loss (48).

In conclusion, present study for the first time showedthat silibinin modulates E-cadherin, b-catenin, Snail, Slug,and Hakai level as well as Akt and Src phosphorylation,which possibly promotes cell–cell contact and inhibitsmigratory and invasive potential of PCA cells. Silibininis nontoxic and widely consumed as a supplement showingits human acceptability, and therefore it is suggested that itshould be studied further in clinically relevant animalmodels to exploit its potential benefits against metastaticPCA in future.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

This work was supported by NCI RO1 grant CA102514.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 10, 2010; revised April 14, 2011; accepted April 18,2011; published online August 4, 2011.

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2011;4:1222-1232. Published OnlineFirst May 5, 2011.Cancer Prev Res Gagan Deep, Subhash Chander Gangar, Chapla Agarwal, et al. Silibinin in Prostate Cancer CellsRole of E-cadherin in Antimigratory and Antiinvasive Efficacy of

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