Apigenin inhibits TGF-β-induced VEGF expression in human prostate carcinoma cells via a Smad2/3- and Src-dependent mechanism

MOLECULAR CARCINOGENESIS

Apigenin Inhibits TGF-b-Induced VEGF Expression inHuman Prostate Carcinoma Cells Via a Smad2/3- andSrc-Dependent Mechanism

Salida Mirzoeva, Carrie A. Franzen, and Jill C. Pelling*

Department of Pathology and the Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, Illinois

Cancer progression relies on establishment of the blood supply necessary for tumor growth and ultimately metasta-sis. Prostate cancer mortality is primarily attributed to development of metastases rather than primary, organ-confined

disease. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis in prostate tissue. Our previousstudies have demonstrated that the chemopreventive bioflavonoid apigenin inhibited hypoxia-induced elevation ofVEGF production at low oxygen conditions characteristic for solid tumors. Low oxygen (hypoxia) and transforming

growth factor-b (TGF-b) are two major factors responsible for increased VEGF secretion. In the present study, experi-ments were performed to investigate the inhibitory effect of apigenin on TGF-b-induced VEGF production and themechanisms underlying this action. Our results demonstrate that VEGF expression is induced by TGF-b1 in human

prostate cancer PC3-M and LNCaP C4-2B cells, and treatment with apigenin markedly decreased VEGF production.Additionally, apigenin inhibited TGF-b1-induced phosphorylation and nuclear translocation of Smad2 and Smad3.Further experiments demonstrated that specific transient knockdown of Smad2 or Smad3 blunted apigenin’s effect onVEGF expression. We also found that apigenin inhibited Src, FAK, and Akt phosphorylation in PC3-M and LNCaP C4-

2B cells. Furthermore, constitutively active Src reversed the inhibitory effect of apigenin on VEGF expression andSmad2/3 phosphorylation. Taken together, our results suggest that apigenin inhibits prostate carcinogenesis by modu-lating TGF-b-activated pathways linked to cancer progression and metastases, in particular the Smad2/3 and Src/FAK/

Akt pathways. These findings provide new insights into molecular pathways targeted by apigenin, and reveal a novelmolecular mechanism underlying the antiangiogenic potential of apigenin. � 2013 Wiley Periodicals, Inc.

Key words: apigenin; TGF-b; Smad2/3; VEGF; prostate cancer

INTRODUCTION

Prostate cancer is the second most common causeof cancer-related deaths in American men. In pros-tate cancer, most deaths are attributed to metastaticdisease rather than primary prostate carcinomas.Solid tumors, such as prostate carcinomas, rely onthe establishment of vascular supply and angiogene-sis for their growth and metastasis. In fact, densityand morphology of tumor microvessels is used as aprognostic biomarker in prostate cancer patientssince it has been reported to be correlated with pros-tate cancer aggressiveness [1,2].

Vascular endothelial growth factor (VEGF) plays acritical role in promoting the formation of bloodvessels, by inducing proliferation, migration, net-work formation, and branching of endothelial cells[3]. In prostate epithelial cells, VEGF represents themost predominantly expressed angiogenic cytokine[4]. VEGF expression is elevated in prostate cancersand VEGF tissue and plasma levels correlate with tu-mor grade and stage [5,6]. VEGF over expression di-rectly correlates with metastatic capability of cells[6–8] and poor prognosis in patients with invasiveprostate cancer [6,9]. Studies have shown that VEGFproduced by tumor cells functions as a growth fac-tor (supporting tumor cell growth) and as an angio-genesis factor (supporting the growth of endothelial

cells). Therefore, suppression of VEGF synthesisand/or inhibition of VEGF activity represent promis-ing approaches to cancer treatment, by disruptingdevelopment and survival of both tumor and

Abbreviations: VEGF, vascular endothelial growth factor; TGF,transforming growth factor; Smad, receptor-regulated Sma- andMad-related protein; PP2, Src kinase inhibitor (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine); DMSO, dimethyl-sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase;RPMI 1640, Roswell Park Memorial Institute; hiFBS, heat-inactivatedfetal bovine serum; PC3-M cells, metastatic human prostate cancercells; LNCaP C4-2B cells, bone metastatic, androgen-refractoryderivative of LNCaP cells; EDTA, ethylenediamine tetraacetic aciddisodium salt; SDS, sodium dodecyl sulfate; PAGE, polyacrylamidegel electrophoresis; TBS-T, Tris-buffered saline with Tween; shRNA,short hairpin RNA; GFP, green fluorescent protein; PBS, phosphatebuffered saline; FAK, focal adhesion kinase; PI3K, phosphatidylinosi-tol 30-kinase; caSrc, constitutively active Src.

Contract grant sponsor: NCI Prostate SPORE; Contract grant num-ber: P50 CA90386; Contract grant sponsor: Zell Foundation; Con-tract grant sponsor: Rosenberg Family Foundation; Contract grantsponsor: Robert H. Lurie Comprehensive Cancer Center; Contractgrant sponsor: ACS post-doctoral fellowship; Contract grant number:PF-10-235-01-CSM.

*Correspondence to: Department of Pathology, Lurie Bldg Room3-115, Northwestern University, 300 E. Superior Street, Chicago, IL60611.

Received 1 June 2012; Revised 28 November 2012; Accepted 17December 2012

DOI 10.1002/mc.22005

Published online in Wiley Online Library(wileyonlinelibrary.com).

� 2013 WILEY PERIODICALS, INC.

endothelial cells, thus inhibiting tumor progressionand metastasis.

VEGF production is regulated by two major mech-anisms, hypoxia (low oxygen supply) and variouscytokines [10,11]. Interplay between hypoxia, cyto-kines, growth, and angiogenic factors determinesdevelopment and progression of cancer [9–11]. Werecently have shown that the chemopreventive bio-flavonoid apigenin (40,5,7-trihydroxyflavone) pre-vents activation of VEGF by hypoxia in prostatecancer cells and inhibits endothelial cell migrationin vitro [12]. Studies in our laboratory and othershave also shown that apigenin is a nontoxic flavonethat exhibits antitumorigenic activity against a vari-ety of cancers [13]. Apigenin fed by gavage wasshown to inhibit prostate tumor xenograft growthin mice [14]. To further delineate anti-cancer prop-erties of apigenin, in the present study we investi-gated whether apigenin affects production of VEGFin response to transforming growth factor-b (TGF-b), a multifunctional cytokine that promotes VEGFproduction in numerous cell lines [15,16].

During normal prostate cell homeostasis, TGF-bregulates prostate growth by inhibiting cell prolifer-ation and inducing apoptosis [11]. In metastaticprostate cancer, however, TGF-b serves as a prostatetumorigenesis promoter [11]. The TGF-b signalingcascade has been implicated in the spread of pros-tate cancer since elevated serum levels of TGF-bwere observed in patients with lymph node and dis-tant site metastases compared to those with local-ized cancer [9,17]. TGF-b signaling is initiated uponbinding of TGF-b to membrane receptors bearingserine/threonine kinase activity, namely TGF-breceptors type I and type II [15,18]. Activation ofTGF-b receptor type I induces phosphorylation of itscytoplasmic effectors, receptor-regulated Sma- andMad-related proteins (Smads). PhosphorylatedSmads associate with mediator proteins and translo-cate to the nucleus [15]. In the nucleus, this hetero-meric Smad complex binds to DNA and regulatestranscription of specific genes, notably VEGF. Block-ing of TGF-b action inhibits tumor viability, tumorcell migration and metastasis in prostate cancer [15–17,19,20]. Therefore, reduction of TGF-b productionand activity may be a promising target for control-ling tumor growth and ultimately prostate cancermetastasis.

We have previously shown that apigenin effec-tively inhibits expression of hypoxia-inducible fac-tor-1a in human prostate cancer cells grown underhypoxic conditions, and affects prostate cancer cellmotility, invasion, and migration via Src/Akt signal-ing [12,21]. In this manuscript, we have investigatedthe effect of apigenin on TGF-b signaling viaSmad2/3 and Src in the highly metastatic humanprostate cancer cell lines PC3-M and LNCaP C4-2B.We have shown that apigenin blocks TGF-b1 activa-tion of Src and Smad2/3 pathways and inhibits

VEGF production. Furthermore, we investigated theeffect of Smad2 and Smad3 knockdown on VEGF ex-pression and demonstrated that specific knockdownof either Smad2 or Smad3 blunted apigenin’s effecton VEGF expression. These results confirmed theimportance of Smad2/3 pathways for apigenin’s in-hibition of TGF-b-induced VEGF expression. Wealso found that constitutively active Src reversed theinhibitory effect of apigenin on Smad2/3 phosphor-ylation and VEGF expression. Apigenin’s ability todownregulate TGF-b1-responsive pathways andVEGF production, in combination with our previousreport that apigenin inhibits vascular cell motility[12], indicates that apigenin may represent an at-tractive candidate compound for prevention of pros-tate cancer metastasis.

MATERIALS AND METHODS

Reagents

All chemicals, protease and phosphatase inhibitorcocktails were from Sigma–Aldrich Co (St. Louis,MO) unless otherwise indicated. Apigenin and Srckinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) were dissolvedin dimethylsulfoxide (DMSO). Recombinant humanTGF-b1 was purchased from R&D Systems (Minne-apolis, MN).

Antibodies

Antibodies to phospho-Smad2 (Ser465/467), phos-pho-Smad3 (Ser423/425), total Smad2, total Smad3,and total Smad2/3 were from Cell Signaling Tech-nologies (Beverly, MA). Antibodies to phospho-Src(Tyr416), phospho-FAK (Tyr576/577), phospho-Akt(Ser473), FAK, and Akt antibodies were from CellSignaling Technologies. Antibodies to total Src andhnRNP A0 were from Santa Cruz Biotechnology(Santa Cruz, CA). Glyceraldehyde-3-phosphate de-hydrogenase (GAPDH) antibody was from Chemi-con International, Inc. (Temecula, CA). HRP-conjugated goat anti-mouse and goat anti-rabbit sec-ondary antibodies were from Bio-Rad Laboratories(Hercules, CA).

Cell Culture

Human prostate cancer PC3-M cells were obtainedfrom Dr. R. C. Bergan (Northwestern University,Feinberg School of Medicine, Chicago, IL) and cul-tured as previously described [12,21]. LNCaP C4-2Bcells, which are a bone metastatic, androgen-refrac-tory derivative of LNCaP cells [22], were a generousgift of Dr. R. S. Taichman (University of Michigan,Ann Arbor, MI). Cells were cultured in Roswell ParkMemorial Institute (RPMI) 1640 (Mediatech, Mana-ssas, VA) containing 10% heat-inactivated fetal bo-vine serum (hiFBS; Invitrogen, Carlsbad, CA), 1 mMglutamine, 100 U/ml penicillin, 100 mg/ml strepto-mycin (Mediatech).

2 MIRZOEVA ET AL.

Molecular Carcinogenesis

Treatment of Cells With TGF-b1

Recombinant human TGF-b1 was reconstituted to2 mg/ml in sterile PBS, aliquoted, and stored at�208C. PC3-M and LNCaP C4-2B cells were grownto 70–80% confluency. To synchronize cellularresponses, cells were serum starved for all experi-ments in RPMI 1640 containing 1 mM glutamine,100 U/ml penicillin, 100 mg/ml streptomycin (Medi-atech) prior to addition of TGF-b1. Cells were serumstarved for 16 h. TGF-b1 was added at a final con-centration of 10 ng/ml in RPMI 1640 containing10% hiFBS, 1 mM glutamine, 100 U/ml penicillin,100 mg/ml streptomycin.

ELISA Measurements of Secreted VEGF

To analyze VEGF secretion, PC3-M and LNCaPC4-2B cells were seeded in 6-well plates, and cul-tured to 80% confluence. Cells were serum starvedfor 16 h and pre-treated with serum-free RPMI 1640medium containing apigenin, PP2 or DMSO (as asolvent control). One hour later, cell culture medi-um containing TGF-b1 along with agents as appro-priate for each experiment was added. Twenty-fourhours later, conditioned media was collected,cleared by centrifugation and stored at �808C.ELISA was performed using the commercial humanVEGF Quantikine kit (R&D Systems) according tocompany protocol. Recombinant human VEGF wasused for calibration. Experiments were carried out atleast two times in triplicates.

MTS Assays

PC3-M and LNCaP C4-2B cells were plated in 96-well plates and cultured to 80% confluence. Cellswere serum starved for 16 h, pre-treated with serum-free RPMI 1640 medium containing apigenin for1 h, then cell culture medium containing TGF-b1was added, along with agents as appropriate foreach experiment. Twenty-four hours later, cell via-bility was assessed by the MTS titer assay accordingto manufacturer’s instructions (Promega, Madison,WI). Formazan concentration (proportional to thenumber of living cells) was quantitated by measur-ing absorbance at 490 nm. Data was expressed as apercent of control where controls were cellstreated with DMSO for the same period of time.Experiments were carried out at least two times intriplicate.

Immunoblotting

Cells were washed with ice-cold PBS and lysed ina buffer containing 20 mM Tris–HCl, 2 mM ethyle-nediamine tetraacetic acid disodium salt (EDTA),10% glycerol, 1% Triton X-100, 150 mM NaCl,protease and phosphatase inhibitor cocktails (pH7.5) for 1 h in the cold room. Lysates were clarifiedby centrifugation at 10 000g for 5 min at 48Cand protein concentrations were determined by

bicinchoninic acid protein assay (Bio-Rad Laborato-ries). Aliquots (40 mg of total protein) were loadedper well and subjected to electrophoresis in 10% so-dium dodecyl sulfate–polyacrylamide gel electro-phoresis (SDS–PAGE) gels (Criterion system, Bio-RadLaboratories) under reducing conditions, followedby transfer to a Protran BA85 0.45 mm nitrocellulosemembrane (Whatman, Sanford, ME). The mem-brane was blocked for 1 h at room temperature in5% non-fat milk in TBS-T (10 mM Tris, 150 mMNaCl, 0.05% Tween 20 (pH 8.0)) and incubated withprimary antibodies in 5% bovine serum albumin inTBS-T overnight at 48C. Primary antibodies wereused at 1:1000 dilution except GAPDH and phos-pho-Src (Tyr-416) antibody which were used at1:80 000 and 1:10 000 dilution, respectively. Immu-noreactive bands were visualized using EnhancedChemiLuminescence detection reagents (Amersham,Piscataway, NJ) and X-ray film (Kodak, Rochester,NY). Digitized X-ray films were quantified usingMultiGauge v2.3 software (Fujifilm Co. Ltd, Tokyo,Japan). Experiments were repeated at least threetimes.

Subcellular Fractionation

PC3-M and LNCaP C4-2B cells were treated withor without TGF-b1 and various agents as describedabove, washed with cold PBS and scraped into hypo-tonic buffer (10 mM HEPES-Na, 10 mM KCl, 1 mMEDTA, 1 mM DTT, 1 mM MgCl2, 5% glycerol,0.1 mM sodium vanadate, 1% protease inhibitorcocktail (pH 7.5)).

Cells were incubated on ice for 10 min and lysedby adding 5 ml of the same buffer containing10% Nonidet P-40 (Sigma-Aldrich Co). Nuclei wereisolated by centrifugation at 1500g for 10 min at48C. Resulting cytoplasmic fractions were frozen at�808C. Nuclei were washed twice by centrifugationat 1500g for 10 min at 48C in buffer containing10 mM HEPES-Na, 10 mM KCl, 1 mM DTT, 1 mMMgCl2 (pH 7.5), and resuspended in buffer contain-ing 20 mM HEPES-Na, 400 mM NaCl, 1 mM EDTA,1 mM DTT, 1 mM MgCl2, 25% glycerol, and prote-ase inhibitors (pH 7.5). Nuclear suspension was kepton ice with frequent vortexing. Fifteen minuteslater, lysed nuclei were clarified by centrifugationat 15 000g for 5 min at 48C, and resulting nuclearfractions were stored at �808C.

Knockdown of Smad2 and Smad3

For Smad2 and Smad3 knockdowns, lentiviralstocks were prepared using short hairpin RNA(shRNA)mir Smad2 (clone V2LHS_251359) andshRNAmir Smad3 (clone V2LHS_215032) from OpenBiosystems (Huntsville, AL). Virus stocks were pre-pared following manufacturer’s protocol. An shRNAnonsilencing GIPZ shRNAmir control vector (OpenBiosystems) was used as a control. PC3-M cells weregrown to about 70% confluency, washed with PBS

APIGENIN INHIBITS TGF-b-INDUCED VEGF EXPRESSION 3


and transduced with corresponding virus in serum-free media. Six hours later, serum-containing mediawas added and cells were incubated for an addition-al 16 h, then conditioned media was replaced by afresh serum-containing media and cells were treatedwith various agents 24 h later.

Adenoviral Cell Transduction

Adenoviral vectors containing either caSrc(Y416F) or green fluorescent protein (GFP) were giftsfrom Dr. Paul Stein and Dr. Kathleen Green (North-western University, Feinberg School of Medicine,Chicago, IL), respectively. PC3-M and LNCaP C4-2Bcells were grown in complete medium to 60–70%confluency, rinsed with PBS and transduced with ei-ther GFP- or caSrc (Y416F) adenovirus in fresh se-rum-containing medium. Cells were gently mixedevery 10 min for 2 h, then adenovirus-containingmedium was aspirated, cells were rinsed with warmPBS and fresh serum-containing medium was added.Cells were incubated for 16 h and treated with vari-ous agents.

Data Analyses

Data were expressed as the mean � standard error(SEM). Statistical significance was evaluated usingPrism software (GraphPad Software, Inc., San Diego,CA). Effects of various treatment conditions werecompared by Student’s t-test. P values of <0.05 wereconsidered statistically significant.

RESULTS

Apigenin Inhibits TGF-b1-Induced VEGF Expression

To examine the effect of apigenin on VEGF ex-pression by human prostate cancer cells, PC3-M andLNCaP C4-2B cells were treated for 1 h in mediumcontaining apigenin (12.5–50 mM) and then wereincubated in the presence or absence of TGF-b1(10 ng/ml) for 24 h. Analyses of VEGF concentra-tion in the conditioned medium of TGF-b1-treatedcells showed a 2.5- and 1.8-fold increase comparedto conditioned medium of vehicle-treated PC3-Mand LNCaP C4-2B cells (P < 0.05), respectively(Figure 1A). Up-regulation of VEGF expression byTGF-b1 was inhibited by apigenin in a dose-depen-dent manner (Figure 1A). VEGF secretion in TGF-b1-treated PC3-M cells was inhibited by 52% and 75%at 25 and 50 mM of apigenin, respectively (P < 0.05;no apigenin vs. apigenin-treated; Figure 1A). VEGFsecretion in TGF-b1-treated LNCaP C4-2B cells wasinhibited by 46% and 60% at 25 and 50 mM of api-genin, respectively (P < 0.05; no apigenin vs. apige-nin-treated; Figure 1A). In view of our previouslypublished findings that apigenin inhibits Src activa-tion [21], we also treated cells with the Src inhibitorPP2, which has been shown to inhibit VEGF produc-tion in a number of cell, including prostate cells[23]. As expected, treatment of cells with 10 mM of

PP2 inhibitor resulted in significant inhibition ofVEGF production: VEGF levels were inhibited by76% in PC3-M cells and 67% in LNCaP C4-2B cells(P < 0.05; no PP2 vs. PP2-treated; Figure 1A). Apige-nin’s effect on VEGF expression was not due to de-creased cell viability as shown by MTS assay(Figure 1B).

Apigenin Inhibits TGF-b1-Induced Activation of Smad2 andSmad3

Induction of VEGF gene expression by TGF-b ismediated by receptor-regulated Smad2 and Smad3[11,18]. Phosphorylation of Smad2 and Smad3 in re-sponse to TGF-b leads to formation of complexeswith mediator Smad4, which are translocated to thenucleus. In the nucleus, Smads associate directlywith specific DNA sequences and transcription fac-tors to regulate expression of target genes. TGF-b/Smad pathways were shown to activate the expres-sion of many downstream genes including VEGF[18].

To delineate the role of Smads in apigenin’s inhib-itory effects on VEGF expression, we assessed wheth-er apigenin affects Smad2/Smad3 activation byTGF-b. We first analyzed apigenin’s effect on phos-phorylation levels of Smad2 and Smad3 proteins toinvestigate the molecular mechanisms underlying

Figure 1. Apigenin down regulates TGF-b1-induced VEGF proteinsecretion by PC3-M and LNCaP C4-2B cells in a dose-dependentmanner. PC3-M and LNCaP C4-2B cells were grown to 80% conflu-ence, serum starved for 16 h and treated with apigenin (12.5, 25,and 50 mM), PP2 (10 mM) or DMSO (as a solvent control) for 1 h.Cells then were incubated in the presence or absence of TGF-b1(10 ng/ml) in serum-containing medium along with other agents asappropriate. (A) After 24 h, conditioned medium was collected, clari-fied by centrifugation and the clarified medium was analyzed byELISA using recombinant human VEGF as a standard. Values are themean from two independent experiments. Asterisk depicts signifi-cantly decreased VEGF concentrations in the conditioned media ofapigenin- or PP2- treated cells compared to that of control (DMSO-treated) cells (P < 0.05). (B) After 24 h, cell viability was assessed byMTS titer assay as described in Materials and Methods Section. Cellviability data were expressed as a percent of control where controlswere the viability of cells treated with DMSO for the same period oftime. Values are the mean from two independent experiments.

4 MIRZOEVA ET AL.


apigenin’s inhibitory effect on VEGF expression.Activated TGF-b receptor type I phosphorylatesreceptor-regulated Smads at their carboxy-termini(Ser465/467 for Smad2 and Ser423/425 for Smad3).We used specific antibodies that recognize thesephosphorylation sites to investigate phosphoryla-tion of Smad2 and Smad3 in TGF-b1-induced cellstreated with apigenin. PC3-M and LNCaP C4-2Bcells were treated for 1 h in medium containing 25or 50 mM of apigenin or 10 mM of PP2 and thenwere incubated in the presence or absence of TGF-b1 (10 ng/ml) for 30 min. Treatment of cells withTGF-b1 led to a rapid increase in phosphorylationlevels of both Smad2 at Ser465/467 and Smad3 atSer423/425 (Figure 2A). Apigenin markedly inhib-ited Smad2 and Smad3 phosphorylation in responseto TGF-b1 (Figure 2A). In contrast, total Smad2 andSmad3 expression levels remain unaffected by apige-nin (Figure 2A). These findings demonstrate thatapigenin specifically affects pathways regulatingSmad2 and Smad3 phosphorylation/activation.

We next investigated whether apigenin affects nu-clear translocation of Smad2 and Smad3 proteins.PC3-M and LNCaP C4-2B cells were treated for 1 hin medium containing 25 or 50 mM of apigenin andthen were incubated in the presence or absence ofTGF-b1 (10 ng/ml) for 1 h. Nuclear fractions wereprepared by hypotonic/Nondet P-40 lysis as de-scribed in Materials and Methods Section. Nuclearfractions were analyzed for total Smad2/Smad3 andhnRNPA0 (a nuclear protein used as a loading con-trol) by specific antibodies. Treatment of PC3-M andLNCaP C4-2B cells with TGF-b1 caused a rapid nu-clear accumulation of Smad2 and Smad3 in controlcells (Figure 2B). Treatment with apigenin at 25 and50 mM markedly attenuated nuclear translocation ofSmad2 and Smad3 proteins (Figure 2B). Since phos-phorylation of Smad2 and Smad3 promotes theirtranslocation to the nucleus, our results obtained inthe Smad2/Smad3 translocation studies (Figure 2B),confirmed our Smad2/Smad3 phosphorylation stud-ies presented in Figure 2A.

Figure 2. Apigenin inhibits activation of Smad2 and Smad3proteins by TGF-b1 in PC3-M and LNCaP C4-2B cells. PC3-M andLNCaP C4-2B cells were cultured to 80% confluency, serum starvedfor 16 h, treated with 25 mM of apigenin (A1), 50 mM of apigenin(A2), PP2 (10 mM), or DMSO (C, as a solvent control) for 1 h, thentreated with TGF-b1 (10 ng/ml) in serum-containing medium alongwith other agents as appropriate. (A) After 30 min, cells werewashed with cold PBS and lysed as described in Materials andMethods Section. Total cell lysates (40 mg total protein/well)were subjected to SDS–PAGE and transferred to nitrocellulosemembrane for immunoblot analysis. Immunoblots were probed withphospho-Smad2 (Ser465/467), phospho-Smad3 (Ser423/425), andGAPDH-specific antibodies. Membranes then were stripped andre-probed with total Smad2 and total Smad3 antibodies. Three

separate experiments were performed with similar results. Immuno-blot shows results from one representative experiment. Bar graphsrepresent the quantification of the phospho-Smads and total Smadsnormalized with the GAPDH bands. (B) PC3-M and LNCaP C4-2Bcells were treated in the absence and presence of TGF-b1 (10 ng/ml)for 1 h. Subcellular fractionation was performed as outlined inMaterials and Methods Section, and equal amounts of proteinfrom cytoplasmic (C) and nuclear (N) fractions were subjected toelectrophoresis in SDS–polyacrylamide gels, followed by Westernblot analysis with antibodies specific for total Smad2, total Smad3,actin (cytoplasmic marker), and hnRNPA0 (nuclear marker). Twoseparate experiments were performed with similar results. Resultsfrom one representative experiment are shown.



Apigenin Inhibits VEGF Expression Via Smad2 and Smad3

Pathways

To confirm more directly that the individualSmad2 and Smad3 pathways are important for inhi-bition of VEGF production by apigenin, we knockeddown Smad2 and Smad3 proteins using lentiviralvectors that express the relevant shRNAmirs vectorsas described in Materials and Methods Section. Con-trol cells were transduced with the nonsilencingshRNAmir construct. Knockdown of Smad2 andSmad3 proteins was confirmed by Western blotanalysis (Figure 3A).

Conditioned media from transfected cells was col-lected for ELISA analysis. Quantitation of VEGF lev-els in the conditioned medium of PC3-M cellsdemonstrated that treatment of control cells withTGF-b1 resulted in a 2.35-fold increase in VEGF pro-duction while Smad2- and Smad3-knockdown cellsproduced only 1.61- and 1.18-fold increase, corre-spondingly (P < 0.05, Smad vs. control shRNA-transduced cells; Figure 3B). Importantly, specificknock-down of either Smad2 or Smad3 proteins sig-nificantly blunted inhibition of VEGF production byapigenin (Figure 3B). Analyses of VEGF concentra-tion in conditioned media of TGF-b1-induced PC3-M cells demonstrated that treatment with apigenin

(50 mM) produced a 68% decrease in VEGF produc-tion by PC3-M cells treated with control construct.In contrast, Smad2- and Smad3-knocked downcells displayed only a 20% and 7% decrease inTGF-b1-induced VEGF levels, respectively (P ¼ NS,control vs. Smad shRNA-transduced cells). Thesedata indicate that apigenin’s ability to inhibit VEGFexpression is mediated by both Smad2 and Smad3pathways.

Apigenin Inhibits TGF-b1-Induced Activation of c-Src

Previous studies from several laboratories haveshown that c-Src, a nonreceptor tyrosine kinase,plays an important role in regulating VEGF expres-sion [24,25]. Furthermore, high expression of Srchas been reported to be associated with elevatedVEGF expression in tumor tissues [26]. On the basisof these reports we examined whether inhibition ofVEGF expression by apigenin in PC3-M and LNCaPC4-2B cells involved c-Src.

The level of phosphorylation of Tyr416 autophos-phorylation site in the activation loop of human c-Src is correlated with its enzymatic activity [27].Therefore, we investigated the activation of Src bymonitoring Tyr416 phosphorylation by immuno-blot analysis (Figure 3A). PC3-M and LNCaP C4-2Bcells were treated for 1 h in medium containing 25or 50 mM of apigenin or 10 mM of PP2 and thenwere incubated in the presence or absence of TGF-b1 (10 ng/ml) for 1 h. Western blot analysis demon-strated that treatment with TGF-b1 led to elevatedphosphorylation of c-Src at Tyr416, and this phos-phorylation was inhibited by apigenin at 25 and50 mM in both PC3-M and LNCaP C4-2B cells(Figure 4A). The induction of Src phosphorylationby TGF-b1 was also blocked in cells treated with10 mM of Src family kinase inhibitor PP2. The levelof total c-Src protein remained unchangedthroughout.

Apigenin Inhibits TGF-b1-Induced Activation c-SrcSubstrates FAK and Akt

To confirm that the observed decrease in level ofc-Src phosphorylation is reflected in decreased c-Srcactivity, we investigated apigenin’s effect on phos-phorylation levels of two downstream substrates ofSrc, focal adhesion kinase (FAK), and Akt [28]. FAKis a well-characterized direct substrate of c-Src. Akt isa distant down-stream target of Src. Src stimulatesAkt activity via the phosphatidylinositol-3 kinase(PI3K) pathway [29]. All three of these kinases, Src,FAK, and Akt, have been shown to play critical rolesin cell survival, apoptosis, tumor development, andmetastasis (for review, see Ref. [30]).

We investigated apigenin’s effect on phosphoryla-tion of a major phosphorylation site of FAK that hasbeen demonstrated to correlate with FAK activity.Tyr576 and Tyr577 of FAK are located in the kinasedomain activation loop and represent a direct

Figure 3. Knockdown of Smad2 or Smad3 proteins in PC3-M cellsaffects induction of VEGF production by TGF-b1 and inhibition byapigenin. PC3-M cells were cultured to about 70% confluency,washed with PBS and infected with either lentiviral shRNA againstSmad2 (Smad2), or Smad3 (Smad3), or nonsilencing shRNA control(C) in serum-free media. Serum-containing media was added 6 h lat-er. Twenty-four hours later, cells were treated with various agents asappropriate. (A) Total cell lysates (40 mg total protein/well) were sub-jected to SDS–PAGE and transferred to nitrocellulose membrane forimmunoblot analysis. Immunoblots were probed with total Smad2,total Smad3, and GAPDH-specific antibodies. Two separate experi-ments were performed with similar results. Immunoblot showsresults from one representative experiment. (B) Conditioned mediumwas collected 24 h after treatment with or without TGF-b1 (10 ng/ml) and apigenin (50 mM), clarified by centrifugation and analyzedby ELISA using recombinant human VEGF as a standard. Values arethe mean from two independent experiments performed in four rep-licates per condition. Asterisk depicts significantly decreased VEGFconcentrations in the conditioned media of apigenin þ TGF-b1-treated cells compared to that of control (DMSO) þ TGF-b1-treatedcells (P < 0.05).

6 MIRZOEVA ET AL.


phosphorylation site for Src family kinases [31].Therefore, we determined if TGF-b1 and/or apigeninaffects the phosphorylation of Tyr576/577 of FAK.PC3-M and LNCaP C4-2B cells were treated for 1 hin medium containing 25 or 50 mM of apigenin or10 mM of PP2 and then were incubated in thepresence or absence of TGF-b1 (10 ng/ml) for 1 h.Immunoblot analysis using phospho-specific anti-bodies revealed that TGF-b1 treatment dramatically

increased the phosphorylation of Tyr576/577 andapigenin inhibited phosphorylation of FAK onTyr576/577 in a dose-dependent manner (Figure 4B).Densitometry analysis indicated that apigenin at25 mM decreased FAK phosphorylation on Tyr576/577 by 72% (PC3-M cells, compare lanes 3 and 2)and 64% (LNCaP C4-2B cells, compare lanes 3and 2). Apigenin at 50 mM decreased FAK phosphor-ylation on Tyr576/577 by 84% (PC3-M cells,

Figure 4. Apigenin inhibits TGF-b1-induced activation of p-Src andits downstream substrates FAK and Akt in PC3-M and LNCaP C4-2Bcells. PC3-M and LNCaP C4-2B cells were treated as described inFigure 2. Cell lysates (40 mg total protein/well) were immunoblottedand probed with phospho-Src (Tyr416) and GAPDH antibodies.Membranes then were stripped and re-probed with total Src

antibodies (panel A). The same lysates were probed for phospho-FAK (Tyr576/577), phospho-Akt (Ser473), and GAPDH with subse-quent re-probing (after stripping the membranes) for total FAK andtotal Akt (panel B). Three separate experiments were performed withsimilar results. Immunoblots show results from one representativeexperiment.



compare lanes 4 and 2) and 95% (LNCaP C4-2Bcells, compare lanes 4 and 2). The level of total FAKprotein remained unchanged throughout.

Akt is a serine/threonine protein kinase regulatedby Src family kinases via the PI3K pathway. PI3K is aheterodimer consisting of two subunits, an 85 kDaregulatory subunit and a 110 kDa catalytic subunit.Phosphorylation of the SH2 domain of PI3K by Srcstimulates its catalytic activity, leading to increasesin intracellular concentration of its products, phos-phatidylinositol (3,4)-bisphosphate and phosphati-dylinositol (3,4,5)-trisphosphate. These productsactivate Akt by directly binding to the pleckstrinhomology domain of the enzyme and via activationof phosphoinositide-dependent protein kinases(reviewed in Ref. [30]). We investigated Akt activityby monitoring Ser473 phosphorylation in lysatesfrom TGF-b1-treated cells with and without apige-nin. PC3-M and LNCaP C4-2B cells were treated for1 h in medium containing 25 or 50 mM of apigeninor 10 mM of PP2 and then were incubated in thepresence or absence of TGF-b1 (10 ng/ml) for 1 h.Immunoblot analysis of cell lysates revealed thatTGF-b1 treatment lead to a substantial increase inAkt phosphorylation on Ser473 (Figure 4B). Apige-nin at 25 mM decreased Akt phosphorylation onSer473 by 40% (PC3-M cells, compare lanes 3 and 2)and 52% (LNCaP C4-2B cells, compare lanes 3 and2). Apigenin at 50 mM decreased Akt phosphoryla-tion on Ser473 by 78% (PC3-M cells, compare lanes4 and 2) and 85% (LNCaP C4-2B cells, comparelanes 4 and 2). The level of total Akt proteinremained unchanged throughout.

Apigenin Inhibits TGF-b1-Induced VEGF Expression andSmad Activation Via c-Src Pathway

Several reports have implicated the Src pathwayin regulation of VEGF production [25,32]. To deter-mine more directly if inhibition of the Src pathwayby apigenin results in inhibition of VEGF produc-tion in PC3-M and LNCaP C4-2B cells, cellswere transduced with adenovirus expressing consti-tutively active Src (caSrc) as described in Materialsand Methods Section. Control cells were transducedwith GFP virus. ELISA quantitation of VEGF levels inthe conditioned medium of apigenin-treated PC3-Mand LNCaP C4-2B cells showed that expression ofcaSrc significantly blunted apigenin’s inhibitoryeffect on VEGF production (Figure 5A). Analyses ofVEGF concentration in conditioned media of TGF-b1-induced PC3-M cells showed that 50 mM of api-genin produced a 70% decrease of VEGF levels incontrol (GFP-transduced) cells. In contrast, apigenintreatment only decreased VEGF levels by 31% inTGF-b1-induced cells which expressed caSrc (P < 0.05,GFP-transduced vs. caSrc-transduced PC3-M cells).Analysis of VEGF concentration in conditioned me-dium of TGF-b1-induced LNCaP C4-2B cells showed

that 50 mM of apigenin produced a 61% decreasein VEGF levels in control (GFP-transduced) cells.In comparison, apigenin treatment only decreasedVEGF levels by 29% in conditioned medium fromTGF-b1-induced cells which expressed caSrc (P < 0.05,GFP-transduced vs. caSrc-transduced LNCaP C4-2Bcells).

To confirm that Src is also important for apigenin’seffect on activation of Smad2/3, we next performedimmunoblot analysis of Smad2 and Smad3 phosphor-ylation levels using PC3-M and LNCaP C4-2B celllysates. As shown in Figure 5B, apigenin dramaticallyinhibited TGF-b1-induced phosphorylation of Smad2and Smad3 in control PC3-M and LNCaP C4-2B cells(transduced with the control GFP-adenovirus). How-ever, apigenin’s ability to inhibit TGF-b1-inducedphosphorylation of Smad2/3 was blocked in cellswhich expressed caSrc (Figure 5B). Taken together,these results indicate that apigenin’s ability to inhibitTGF-b1-induced Smad2/3 activation and subsequentVEGF expression is mediated by Src.

DISCUSSION

This study is the first to demonstrate that plantflavonoid apigenin inhibits VEGF production byprostate cancer cells via both Smad- and non-Smad-dependent mechanisms.

We have demonstrated in this report that treat-ment of prostate cancer cells with TGF-b1 activatedSmad2 and Smad3 transcription factors, and apigenininhibited this activation. To the best of our knowl-edge, this is the first report to demonstrate that apige-nin inhibits TGF-b-induced activation of Smad2 andSmad3. We performed transfection studies, using len-tiviral shRNAmir constructs, to establish if either ofthese so-called co-Smads were involved in the regula-tion of VEGF expression by apigenin. Our studyrevealed that individual silencing of Smad2 or Smad3proteins significantly diminished VEGF productionby PC3-M cells. Notably, silencing of Smad3 led to agreater decrease in both basal and TGF-b1-inducedVEGF production as compared to silencing of Smad2,suggesting greater importance of Smad3 for VEGFproduction in PC3-M cells. Similar findings werereported for other cell types [33,34].

Most importantly, we have also demonstratedthat both Smad2 and Smad3 are essential for regula-tion of VEGF production by apigenin. We observeda significantly diminished response to apigenintreatment in Smad2- and Smad3-knockdown cells,compared to the control vector-transduced cells. Wedemonstrated that knockdown of either Smad2 orSmad3 protein was sufficient to abolish the inhibi-tion of VEGF production by apigenin.

We also have shown in this study that TGF-b1treatment activated the Src pathway and apigenininhibited this effect in a dose-dependent manner.Furthermore, expression of caSrc blunted apigenin’s

8 MIRZOEVA ET AL.


effect on Smad2/3 activation, confirming that theSrc pathway is key for apigenin action. Most impor-tantly, we found that apigenin inhibited the TGF-b1-induced increase in VEGF production by prostatecancer cells, and expression of caSrc abrogated api-genin’s effect. Taken together, these findings illus-trate the importance of Smad2, Smad3, and Srcpathways in apigenin’s chemopreventive activity inprostate cancer.

Formation of new blood vessels, or angiogenesis,plays a critical role in tumor progression. Angiogene-sis inhibitors that suppress the formation of newblood vessels have emerged recently as a new class ofdrugs for cancer treatment [35]. Preclinical studiesusing anti-VEGF antibodies or inhibitors targeted toVEGF receptors have been shown to suppress prostatecancer growth in vivo [3,36]. These studies demon-strate that VEGF is critical for cancer growth and that

blocking VEGF is an effective strategy for treatingprostate cancer. Thus, there is tremendous interest indeveloping novel therapeutic agents that regulateVEGF expression and signaling.

In addition, a great deal of research effort is aimedat identifying chemopreventive agents that possessanti-angiogenic capabilities. While several of theangiogenesis inhibitors are currently being testedin clinical trials for chemotherapeutic agent devel-opment [37,38], dietary-based anti-angiogenesisapproaches represent an attractive strategy in cancerchemoprevention. A major merit to this approach isthat dietary agents have in large part proven to besafe for human use.

Flavonoids comprise a significant component ofthe human diet; flavonoid intake from a normaldiet is estimated to be 1 g/day [39]. Apigenin is anabundant flavonoid present in fruits and vegetables

Figure 5. Transduction of constitutively active Src renders PC3-Mand LNCaP C4-2B cells less sensitive to apigenin. PC3-M and LNCaPC4-2B cells were transduced with GFP- or caSrc-adenovirus asdescribed in Materials and Methods Section. After serum starvationfor 16 h, cells were pre-treated with 12.5–50 mM of apigenin,10 mM of PP2, or DMSO for 1 h, then treated in the presence orabsence of 10 ng/ml of TGF-b1 in serum-containing medium. (A)Twenty-four hours later, conditioned medium was collected, clarifiedby centrifugation, and the VEGF concentration in the clarified mediumwas analyzed by ELISA using recombinant human VEGF as astandard. Values are the mean from two independent experiments.

Asterisk depicts significantly increased VEGF concentrations in theconditioned media of caSrc-transduced cells compared to that ofcontrol (GFP-transduced) cells (P < 0.05). (B) Thirty minutes later,cells were washed with cold PBS and lysed. Forty micrograms of totalprotein was loaded per well, subjected to SDS–PAGE and transferredto nitrocellulose membrane. Immunoblots were probed with GAPDH,phospho-Smad2 (Ser465/467)- and phospho-Smad3 (Ser423/425)-specific antibodies. Membranes then were stripped and re-probedwith total Smad2 and total Smad3 antibodies. Two separate experi-ments were performed with similar results. Immunoblot presentsresults from one representative experiment.



such as parsley, cherries, grapes, onions, and broc-coli [37]. Animal studies have shown that dietaryadministration of apigenin to mice can result intissue concentrations ranging from 1.5 to 86 mM[40]. Thus tissue partitioning evidence for apigeninis encouraging for chemopreventive strategies, andsuggests our dosing levels would be effective forinhibition of both TGF-b activated Smad2/3 andSrc pathways as shown herein in our cell-basedstudies.

Studies in numerous laboratories show that api-genin exhibits chemopreventive activity in skin[14,41], breast [14], ovarian [42], lung, colon [14],and prostate [12,21,43,44] cancer cells. However,the molecular mechanisms for apigenin’s chemo-preventive activity are not fully understood. Ourlaboratory has reported previously that apigenindecreased cell motility and invasiveness in humanprostate carcinoma cells [21]. This quality of apige-nin is thought to be important for its chemo-preventive effect since it is anticipated thatdecreasing cell motility and invasiveness would beassociated with reduced cancer spreading and me-tastases. We have shown previously that apigenininhibits basal as well as hypoxia-induced VEGF lev-els in human prostate cancer cells [12]. In the pres-ent report we demonstrate in cell-based studiesthat apigenin inhibits TGF-b1-induced VEGF ex-pression in human metastatic prostate carcinomacell lines via Smad2/Smad3- and Src-dependentmechanisms. Apigenin inhibited TGF-b-activatedVEGF expression and the Smad2/Smad3 and Src/FAK/Akt pathways, two pathways linked to angio-genesis and metastasis.

In our recent report [12] and in our studies pre-sented herein, we investigated apigenin’s effects onthe major pathways that regulate expression ofVEGF, a key angiogenesis factor. The mechanismsthat regulate VEGF production have been the focusof intensive investigations over the past decade innumerous laboratories (reviewed in Ref. [14]). Twomajor mechanisms that regulate VEGF productionby prostate cancer cells have been identified, name-ly, the hypoxia-regulated signaling pathways andthe TGF-b signaling pathways. Hypoxia is known tobe a hallmark feature of solid tumors resulting inincreased angiogenesis. Hypoxia pathways are acti-vated in many cancers and are amplified by a widevariety of oncogenic pathways and growth factors,such as TGF-b. Both TGF-b and hypoxia pathwayssynergize in the regulation of VEGF gene expressionat the transcriptional level [10]. We previouslyreported that apigenin efficiently blocks the signal-ing pathways that are triggered in prostate cancercells in response to hypoxia. Apigenin treatment re-duced the stability of hypoxia-inducible factor-1a

and it blocked the hypoxia-induced increase inVEGF production [12]. In our present report, our in-vestigative efforts with apigenin have focused on

targeting the pathways that prostate cancer cells usein mounting the response to TGF-b. Using highlymetastatic human prostate cancer PC3-M andLNCaP C4-2B cell lines, we discovered that apigenineffectively intercepts TGF-b1 signaling to down-stream effectors Smad2 and Smad3 and blocks theTGF-b1-induced increase in VEGF expression. Thesefindings provide, to the best of our knowledge, thefirst demonstration of apigenin’s ability to down-regulate TGF-b1 signaling.

Additionally, in our results presented here wedemonstrated that apigenin is a potent inhibitor ofTGF-b-activated Src. Src activity has been implicatedin cancer progression and the regulation of cancercell migration [45]. Src activity also plays a criticalrole in the tumor microenvironment, promotingexpression of pro-angiogenic factors [44]. In endo-thelial cells, Src is activated by VEGF, resulting inincreased vascular permeability and neovasculariza-tion [46]. Furthermore, Src signaling might also beinvolved in prostate tumor metastasis to bone; Srcfamily kinases are involved in signaling of osteo-clasts [47].

We have demonstrated in the present study thatTGF-b1 treatment results in a transient increase inphosphorylation levels of Src and its downstreamsubstrates, FAK and Akt. Since it has been reportedthat Src signaling plays an important role in TGF-baction [48–50], we investigated whether Src is im-portant for inhibition of Smad2/3 activation andVEGF production by apigenin. Apigenin has beenpreviously shown by our laboratory to down-regu-late basal Src activity in the prostate cancer PC3-Mcell line [21]. In the present study, we showed thatapigenin inhibited TGF-b1-induced activation ofSrc. Using adenovirus expressing caSrc, we haveshown that caSrc reversed apigenin’s inhibition ofTGF-b1 activation of Smad2/Smad3 and productionof VEGF, supporting our hypothesis that apigenininhibits VEGF expression by modulating the Srcpathway. Although our results indicate that apige-nin can suppress VEGF expression via the Smad/Srcaxis, our data do not rule out the possibility thatapigenin may modulate other pathways that arealso important for regulating VEGF expression.

Interestingly, the expression of caSrc did not havean effect on PP2’s inhibition of VEGF secretion inTGFb1-induced cells (Figure 5A). This observationbears significance in light of studies by Maeda et al.[51] demonstrating that the widely used Src inhibi-tors PP2 and PP1 also are potent inhibitors of TGF-breceptor kinase activity.

Our results demonstrate that the ability of TGF-b1to induce VEGF expression was significantly inhib-ited by apigenin in a dose-responsive manner.Apigenin’s inhibitory effect on hypoxia- and TGF-b1-induced activation of VEGF expression ([12] andthis paper), in conjunction with its direct inhibitoryeffect on human endothelial cell migration [12],

10 MIRZOEVA ET AL.


provides evidence that apigenin has multi-facetedanti-angiogenic capabilities.

In summary, in this study we have shown thatapigenin inhibits TGF-b1-induced VEGF expressionin human prostate cancer cells. Our results furtherdemonstrate that the mechanisms underlying api-genin’s action include its ability to inhibit bothSmad- (Smad 2 and Smad3) and non-Smad (Src/FAK/Akt) activation. This finding adds new insightinto the potential mechanism underlying the anti-angiogenic action of apigenin. Development ofnovel pharmacological intervention against aber-rant TGF-b signaling may potentially inhibit pros-tate cancer spread and decrease the morbidityassociated with development of metastases in pros-tate cancer patients. These results would help inthe design of future strategies of developing apige-nin as a potential chemopreventive agent whenused alone or in combination with current antican-cer drugs.

ACKNOWLEDGMENTS

This work was supported in part by NCI ProstateSPORE grant P50 CA90386 (pilot funding to J.C.P.),by the Zell Foundation (J.C.P. is a Zell Scholar), bysupport from the Rosenberg Family Foundation, bythe Robert H. Lurie Comprehensive Cancer Center,and by ACS post-doctoral fellowship grant PF-10-235-01-CSM (C.F.).

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Apigenin inhibits TGF-β-induced VEGF expression in human prostate carcinoma cells via a Smad2/3- and Src-dependent mechanism