Cytokine 56 (2011) 403–410

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Cytokine

journal homepage: www.elsevier .com/locate / issn/10434666

Modulation of YY1 and p53 expression by transforming growth factor-b3 in prostatecell lines

Silvia Caggia, Massimo Libra, Grazia Malaponte, Venera Cardile ⇑Department of Bio-Medical Sciences, University of Catania, V.le A. Doria 6, 95125 Catania, Italy

a r t i c l e i n f o

Article history:Received 18 March 2011Received in revised form 27 June 2011Accepted 28 June 2011Available online 31 July 2011

Keywords:ApoptosisCancerNOPI3K/AktTGF-b3

1043-4666/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.cyto.2011.06.024

⇑ Corresponding author. Address: Department of Bogy), University of Catania, V.le A. Doria 6, 951257384040; fax: +39 095 7384217.

E-mail addresses: silviacaggia@alice.it (S. Caggia)gmalaponte@unict.it (G. Malaponte), cardile@unict.it

a b s t r a c t

Transforming growth factor-b (TGF-b) is the prototype of a family of secreted polypeptide growth factors.These cytokines play very important roles during development, as well as in normal physiological anddisease processes, by regulating a wide array of cellular processes, such as cell growth, differentiation,migration, apoptosis, and extracellular matrix production. TGF-b utilizes a multitude of intracellular sig-nalling pathways in addition to Smads with actions that are dependent on circumstances, including dose,target cell type, and context. The aims of this research were (i) to verify the effects of dose-dependentTGF-b3 treatment on YY1 and p53 expression, in BPH-1 cell line, human benign prostate hyperplasia,and two prostate cancer cell lines, LNCaP, which is androgen-sensitive, and DU-145, which is andro-gen-non responsive, (ii) establish a correlation between p53 and YY1 and (iii) determine the expressionof a number of important intracellular signalling pathways in TGF-b3-treated prostate cell lines. Theexpression of YY1, p53, PI3K, AKT, pAKT, PTEN, Bcl-2, Bax, and iNOS was evaluated through Western blotanalysis on BPH-1, LNCaP, and DU-145 cultures treated with 10 and 50 ng/ml of TGF-b3 for 24 h. The pro-duction of nitric oxide (NO) was determined by Griess reagent and cell viability through MTT assay. Theresults of this research demonstrated profound differences in the responses of the BPH-1, LNCaP, and DU-145 cell lines to TGF-b3 stimulation. We believe that the findings could be important because of the clin-ical relevance that they may assume and the therapeutic implications for TGF-b treatment of prostatecancer.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Transforming growth factor family members are multifunc-tional cytokines that control a diverse array of cellular processesincluding cell proliferation, morphogenesis, migration, extracellu-lar matrix production, cytokine secretion, and apoptosis [1]. Trans-forming growth factor-b (TGF-b) is a pleiotropic growth factor withactions that are dependent on circumstances, including dose, tar-get cell type, and context. TGF-b can elicit both growth-promotingand growth-suppressive activities. In normal tissues, TGF-b gener-ally acts to restrict growth and maintain differentiation. However,during tumorigenesis, changes in TGF-b expression and cellular re-sponses can promote tumorigenesis. TGF-b pathway has beenimplicated in cancer and has been recently considered as a puta-tive therapeutic target. TGF-b ligands act through transmembranereceptors to activate several downstream signal transduction path-

ll rights reserved.

io-Medical Sciences (Physiol-Catania, Italy. Tel.: +39 095

, mlibra@unict.it (M. Libra),(V. Cardile).

ways [2]. The Smad pathway was the first signalling pathway iden-tified to mediate TGF-b effects and remains the best characterized.More recently, multiple non-Smad pathways have been implicatedin mediating TGF-b effects downstream of the receptors. Theirinvolvement in the changing responses of cells to TGF-b are justbeginning to be probed [3,4]. Studies in mammary epithelial cellshave shown that TGF-b promotes motility through mechanismsindependent of Smad signalling, possibly involving activation ofthe phosphatidylinositol 3-kinase (PI3K)/Akt and/or mitogen-acti-vated protein kinase (MAPK) pathways [5–7]. However, the com-plexity of the role of TGF-b in cancer biology involving aspects oftumour suppression and tumour progression requires a thoroughunderstanding of the TGF-b function [8].

Yin Yang 1 (YY1, also known as d, NF-E1, and UCRBP) is a mul-tifunctional nuclear protein that acts as a repressor, an activator oreven an initiator of transcription. Inspired by its dual transcrip-tional activity, Shi et al. [9] named the protein ‘‘Yin Yang 1’’ fromthe Chinese ‘‘Yin’’, for repression and ‘‘Yang’’ for activation. It ishighly conserved between species and is ubiquitously expressedin different tissues including brain, heart, limb, and immune sys-tem [10] and, certainly in the mouse, is essential for embryo viabil-ity [11]. Human YY1 is a protein composed of 414 amino acids witha calculated molecular weight of 44 KDa [12,13]. It is involved in

404 S. Caggia et al. / Cytokine 56 (2011) 403–410

the transcriptional control of a large number of mammalian genes,approximately 10% of the total mammalian gene set [14]. Thesefeatures suggest that YY1 might have an important role in cell biol-ogy, including cell cycle, control, embryogenesis, viral infection,programmed cell death, oncogenesis, but its role is quite contro-versial and also dependent on specific cell type. Previous studiesidentified a plethora of potential YY1 target genes, the productsof which are important for proliferation and differentiation [15].YY1 has been found to be associated with the tumour suppressorp53, that plays a crucial role in the cellular response to genotoxicstress and mediates either growth arrest or apoptosis, dependingon cellular conditions [16]. Inactivation of the p53 gene is a keyevent in the induction of malignant transformation in majority ofhuman tumours [17]. It controls a number of important eventsand among these the best documented are cell cycle [18], apopto-sis [19], differentiation and development [20]. Each of these biolog-ical roles of p53 contributes to the ability of p53 to limit thetumorigenicity of cells. The p53 protein is also implicated in a spin-dle checkpoint [21] and in the induction of either differentiation[22] or senescence [23]. p53 is a transcription factor that binds ina sequence-specific manner to target sites in gene promoters andactivates transcription of these genes [13], but the regulation ofp53 gene itself is not that well understood. Yakovleva et al. [13]showed that YY1 inhibits p53-activated transcription from thep53-binding site that contains the ACAT sequence. A protectiverole of YY1 from apoptosis was suggested by studies using siRNAor genetic-targeted mutation in lymphoid cells [24]. It was sug-gested that YY1 exerts anti-apoptotic functions by negatively reg-ulating Hdm2-mediated p53 degradation. However, knock-downexperiments of yy1 in embryonic carcinoma cell line did not in-duce apoptosis, despite increased p53 levels [25]. In addition, stud-ies in mouse embryonic fibroblasts [26] or in oligodendrocytelineage cells [27] indicated that decreased level of YY1 did not af-fect p53 levels or increase apoptosis. Seligson et al. [28] detectedYY1 overexpression in a large series of prostate cancer and pros-tatic intraepithelial neoplasia compared to normal or benign pros-tatic hypertrophy tissues.

Thus, in this research, in BPH-1 cell line, human benign prostatehyperplasia, and two prostate cancer cell lines, LNCaP, which isandrogen-sensitive, and DU-145, which is androgen-non respon-sive, an attempt has been made to verify the effects of dose-depen-dent TGF-b3 treatment on YY1 and p53 expression, to know thebinding of YY1 to p53 and establish a correlation between p53and YY1, and determine the expression of a number of importantintracellular signalling pathways in TGF-b3-treated prostate celllines.

2. Materials and methods

2.1. Cell cultures and treatments

Human benign prostate hyperplasia BPH-1, human prostatecancer androgen-non responsive DU-145 and androgen-responsiveLNCaP cells were purchased from the American Type CultureCollection. The BPH-1 and LNCaP cell lines were grown in RPMI-1640 medium supplemented with 10% foetal calf serum, 1 mMglutamine and 10 ll/ml penicillin–streptomycin. Androgen-nonresponsive DU-145 human prostate cancer cells were maintainedin Earle minimal essential medium (EMEM), containing 10% foetalcalf serum, 1 mM L-glutamine, antibiotics (50 IU/ml penicillin and50 lg/ml streptomycin) and 1% non essential amino acids. The cul-tures were incubated at 37 �C in humidified 5% CO2/95% atmo-sphere and transferred to subcultures every 3 days followingtreatment with trypsin–EDTA. The cells were treated 1 day beforethey reached confluence. For experiments, 24 h before the cells

were trypsinized, counted in a haemocytometer, and plated in 96wells plates (8 � 103/200 ll/microwell for MTT and nitric oxidedetermination) and in 100 mm Petri-dishes (1.5 � 106/10 ml/wellfor Western blot). Each experimental prostate cell line was treatedor not (untreated controls) with 10 or 50 ng/ml of TGF-b3 (GRF-10696; Immunological Sciences). After 24 h each sample wastested for expression of YY1, p53, PI3K, AKT, pAKT, PTEN, Bcl-2,Bax and iNOS, cell viability and production of nitric oxide. To assessif YY1 activity is essential for p53 expression in prostate cell, wecarried out target-specific YY1 knockdown using RNA interferencetechniques by YY1-specific siRNAs. The sequences of the siRNAconstructs used for this study are as follows: GENE 1S1: 50-GAACUCACCUCCUGAUUA-U55-30; GENE 1AS1: 50-AUAAUCAGGAGGUGAGUU-C55-30) (Eurogentec). These constructs were transfectedinto cells using the transfection METAFECTENE� SI (Cat. T100-1.0,Biontex) reagent according to the manufacturer’s protocol. Briefly,we seeded 2 ml of the experimental prostate cancer cell linessuspension in complete cell culture medium at a density of5 � 105 cells/ml in 6-well plates. RNA and mix for transfection di-luted according to the manufacturer’s instructions were left toincubate for 24 h in presence and absence of TGF-b3 at the concen-tration of 10 and 50 ng/ml. At the end of this time, cells werecollected and Western blot assay performed.

2.2. Western blot analysis

The expression of YY1, p53, PI3K, AKT, pAKT, PTEN, Bcl-2, Bax,and iNOS was evaluated by Western blot analysis. Briefly, the un-treated and TGF-b3-treated cell lines were washed twice withice-cold PBS and collected with lysing buffer (10 mM Tris–HCl plus10 mM KCl, 2 mM MgCl2, 0.6 mM PMSF, and 1 % SDS, pH 7.4). Aftercooling for 30 min at 0� C, the cells were sonicated. Twenty micro-grams of total protein, present in the supernatant, were loaded oneach lane and separated by 4–12% Novex Bis–Tris gel electrophore-sis (NuPAGE, Invitrogen). Proteins were then transferred to nitro-cellulose membranes (Invitrogen) in a wet system. The transferof proteins was verified by staining the nitrocellulose membraneswith Ponceau S and the Novex Bis–Tris gel with Brillant blue R.Membranes were blocked in Tris buffered saline containing 0.01%Tween-20 (TBST) and 5% non-fat dry milk at 4 �C overnight. Rabbitpolyclonal anti-YY1 (H-414; sc-1703, Santa Cruz Biotechnology,Santa Cruz, CA) (1:300 dilution), -p53 (FL-393; sc-6243, Santa CruzBiotechnology, Santa Cruz, CA) (1:300 dilution), -PI3K (PI 3-kinasep85a (B-9) sc-1637, Santa Cruz Biotechnology) (1:200 dilution), -AKT (#9272, Cell Signaling) (1:1000 dilution), -pAKT (Ser473,193H12; #4058, Cell Signaling) (1:1000 dilution), -PTEN(WH0005728M1, Sigma–Aldrich) (1:1000 dilution); -Bcl-2(SAB2500154, Sigma Aldrich) (1:500 dilution), -Bax (B3428, Sig-ma–Aldrich) (1:2000 dilution), NOS2 (N-20, sc-651, Santa Cruz Bio-technology) (1:300 dilution), and -actin (A2066; Sigma–Aldrich)(1:5000 dilution) antibodies were diluted in TBST and membranesincubated for 2 h at room temperature. Antibodies were detectedwith horseradish peroxidase-conjugated secondary antibody usingthe enhanced chemiluminescence detection Supersignal West PicoChemiluminescent Substrate (Pierce Chemical Co., Rockford, IL).Bands were measured densitometrically and their relative densitycalculated based on the density of the actin bands in each sample.Values were expressed as arbitrary densitometric units (A.D.U.)corresponding to signal intensity.

2.3. Cell viability

The MTT proliferation assay is based on the conversion by mito-chondrial dehydrogenases of a substrate containing a tetrazoliumring into blue formazan, detectable spectrophotometrically. Thelevel of blue formazan is then used as an indirect index of cell

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index. Briefly, cell cultures (8 � 103 cells/microwell) were set up inflat-bottomed 200 ll microplates, incubated at 37 �C in a humidi-fied atmosphere of 95% air/5% CO2 and after 24 h (60–70% conflu-ence) treated with 10 and 50 ng/ml of TGF-b3 for 24 h. Four hoursbefore the end of the culture period, 20 ll of 0.5% MTT in PBS wasadded to each microwell. After incubation with the reagent, thesupernatant was removed and replaced with 100 ll of DMSO.The optical density of each sample was measured using a micro-plate spectrophotometer (Titertek Multiskan; DAS) at k = 550 nm.Each sample was tested in quadruplicate (n = 12).

2.4. Determination of nitrite levels

Nitrite was determined by adding 100 ll of Griess reagent (1%sulphanylamide and 0.1% naphthyl-ethylenediamine dihydrochlo-ride in 5% of hydrochloric acid) to 100 ll of samples. The opticaldensity at k = 550 nm was measured using a microtitre plate reader(Titertek Multiskan; DAS). Nitrite concentrations were calculatedby comparison with respective optical densities of standard solu-tions of sodium nitrite prepared in medium.

2.5. Statistical analysis

Each experiment was repeated at least three times in triplicateand the mean ± SEM for each value was calculated. Statistical anal-ysis of results [Student’s t-test for paired and unpaired data; vari-ance analysis (ANOVA)] was performed using the statisticalsoftware package SYSTAT, version 9 (Systat Inc., Evanston IL,USA). A difference was considered significant at P < 0.05.

3. Results

Three human prostate cell lines, namely, the cancer androgen-dependent LNCaP, the cancer androgen-independent DU-145, andthe benign hyperplasia BPH-1 were treated with 10 and 50 ng/mlof TGF-b3 for 24 h. The results of the MTT assay indicated thatTGF-b3 has no effect on viability of LNCaP, because treatment ofthese cells with 10 and 50 ng/ml did not reduce their ability tometabolize tetrazolium salts (Fig. 1). On the contrary, TGF-b3 in-creased and decreased the cell viability of BPH-1 and DU-145,respectively, particularly at 50 ng/ml.

To determine whether the difference between the cell responseto TGF-b3 resulted from an alteration of downstream signal trans-duction pathways resulting from changes in expression of some pro-

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Fig. 1. BPH-1, human benign prostate hyperplasia, LNCaP, androgen-sensitive, andDU-145, androgen-non responsive, cell viability measured with tetrazolium saltassay (MTT) after 24 h of treatment with TGF-b3 (10 and 50 ng/ml). The values ofoptical density measured at k = 550 nm are reported as percentage with respect tothe optical density registered for untreated control (CTRL), the latter considered as100 % of cell viability. The values are the mean ± SEM of three experimentsperformed in quadruplicate. ⁄p < 0.05 compared to untreated BPH-1 (CTRL).�p < 0.05 compared to untreated DU-145 (CTRL).

teins that modulate signalling, we examined, by Western blot asdescribed in Section 2, the expression of a number of importantintracellular signalling pathways in response to TGF-b3 stimulation.

As shown in Fig. 2A, Western blot analysis revealed a dose-dependent increase in iNOS levels following TGF-b3-treatment inDU-145 cells. The data showed that untreated and TGF-b3-treatedBPH-1 did not express iNOS, untreated LNCaP produced higher andlower quantity of iNOS with respect to BPH-1 and DU-145, respec-tively, confirming the disparities in RNS generation in normal cellscompared to cancer cells. After 24 h, iNOS levels in treated-DU-145cells had increased around twofold at 10 ng/ml, with further in-creases (about fourfold)) at 50 ng/ml. Compared to the untreatedcontrols, TGF-b3 did not modify the levels of iNOS in BPH-1- andLNCaP-treated cells both at 10 and 50 ng/ml. It may be noted thatin prostate cultures NO production has the same behaviour as theexpression of iNOS (Fig. 2B).

Moreover, because Smad proteins transduce TGF-b signals thatregulate cell growth and differentiation, and YY1 has been identi-fied as a transcription factor that positively or negatively regulatestranscription of many genes, as a novel Smad-interacting protein,the cells were analyzed for YY1, and p53 expression. The findingsdemonstrate that compared to the untreated cells in BPH-1 cellsTGF-b3-treatment resulted in dose-dependent increased expres-sion of YY1 (threefold at 50 ng/ml) (Fig. 3) and decreased modifica-tion of p53 (twofold at 50 ng/ml) (Fig. 4); TGF-b3 in LNCaP did not

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Fig. 2. Effects of TGF-b3 on (A) iNOS expression and (B) nitric oxide (NO)production in BPH-1, LNCaP and DU-145 determined by Western blot and Griessreaction, respectively. Experimental cells were treated with 10 and 50 ng/ml for24 h. Data show the relative expression (mean + SEM) of calculated as arbitrarydensitometric units (A.D.U.) and lM collected from three independent experiments.⁄p < 0.05 with respect to untreated DU-145.

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Fig. 3. Effects of TGF-b3 on YY1 expression in BPH-1, LNCaP and DU-145determined by Western blot. Experimental cells were treated with 10 and 50 ng/ml for 24 h. Data show the relative expression (mean + SEM) of calculated asarbitrary densitometric units (A.D.U.) collected from three independent experi-ments. � and � denote significant differences of values compared to untreatedcontrol. ⁄p < 0.05 compared to untreated BPH-1 (CTRL). �p < 0.05 with respect tountreated DU-145.

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Fig. 4. Effects of TGF-b3 on p53 expression in BPH-1, LNCaP and DU-145determined by Western blot. Experimental cells were treated with 10 and 50 ng/ml for 24 h. Data show the relative expression (mean + SEM) of calculated asarbitrary densitometric units (A.D.U.) collected from three independent experi-ments. ⁄p < 0.05 compared to untreated BPH-1 (CTRL). �p < 0.05 with respect tountreated DU-145.

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Fig. 5. Results of YY1 knockdown using RNA interference techniques by YY1-specific siRNAs determined by Western blot in (A) BPH-1, (B) LNCaP and (C) DU-145. The sequences of the siRNA constructs used for this study are as follows: GENE1S1: 50-GAACUCACCUCCUGAUUA-U55-30; GENE 1AS1: 50-AUAAUCAGGAGGUGAG-UU-C55-30 . The constructs were transfected into cells using the transfectionMETAFECTENE� SI. Data show the relative expression (mean + SEM) of calculatedas arbitrary densitometric units (A.D.U.) collected from three independent exper-iments. (A) ⁄p < 0.05 (YY1) compared to untreated BPH-1 (CTRL). >p < 0.05 (p53)compared to untreated BPH-1 (CTRL). (B) �p < 0.05 (YY1) compared to untreatedLNCaP (CTRL). jp < 0.05 (p53) compared to untreated LNCaP (CTRL). (C) sp < 0.05(YY1) with respect to untreated DU-145. dp < 0.05 (p53) with respect to untreatedDU-145.

406 S. Caggia et al. / Cytokine 56 (2011) 403–410

significantly modify the expression of YY1 (Fig. 3) and p53 (Fig. 4);it in DU-145 dose-dependently inhibited expression of YY1 (three-fold at 50 ng/ml) in parallel increasing p53 (twofold) (Figs. 3 and4). The protein extracts prepared from prostate cell lines werenot transfected or were transfected with a scramble siRNA controlor YY1 siRNA construct and analyzed with Western blot usingpolyclonal antibodies against YY1, p53, and b-actin. As summa-rized in Fig. 5, Western blot analyses indicated up 85% reductionin the YY1 protein level in transiently transfected BPH-1 (whereTGF-b3-treatment resulted in dose-dependent increased expres-sion of YY1), LNCaP and DU-145 cells, while control cells with notransfection and with transfection using another siRNA construct

containing a scrambled sequence showed no change in the YY1protein level. Two independent Western blots using b-actin andp53 antibodies also confirmed the target-specific knockdown ofYY1 by this siRNA construct (Fig. 5).

Several findings suggest a role of PI3K in TGF-b3 signalling. TGF-b3 can rapidly activate PI3K, as indicated by the phosphorylation ofits downstream effector Akt [29]. Other studies suggest a negativeregulation of the PI3K/AKT pathway by p53 through the transcrip-tional activation of PTEN [30]. PTEN antagonizes PI3K function bydephosphorylating phosphoinositol triphosphate (PIP3), resultingin the reduction in the phosphorylated AKT fraction and G1 arrest[31]. Thus, we determined the expression of PI3K, Akt, pAkt, andPTEN in the experimental cultures. The results show that TGF-b3induced a dose-dependent increase of PI3K in BPH-1 (+100% at50 ng/ml compared to the respective untreated controls), a not sig-nificant increase in LNCaP, and a dose-dependent decrease in DU-145 (�80% at 50 ng/ml with respect to the untreated controls)(Fig. 6). The results of the levels of AKT and pAKT have been sum-marized in Fig. 7. Compared to untreated cells, there was an in-crease dose-dependent of AKT and pAKT in BPH-1; no change ofAKT and pAKT in LNCaP; a decrease dose-dependent of AKT and

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Fig. 6. Effects of TGF-b3 on PI3K expression in BPH-1, LNCaP and DU-145determined by Western blot. Experimental cells were treated with 10 and 50 ng/ml for 24 h. Data show the relative expression (mean + SEM) of calculated asarbitrary densitometric units (A.D.U.) collected from three independent experi-ments. ⁄p < 0.05 compared to untreated BPH-1 (CTRL). �p < 0.05 with respect tountreated DU-145.

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Fig. 7. Effects of TGF-b3 on Akt/pAkt expression in BPH-1, LNCaP and DU-145determined by Western blot. Experimental cells were treated with 10 and 50 ng/mlfor 24 h. Data show the relative expression (mean + SEM) of calculated as arbitrarydensitometric units (A.D.U.) collected from three independent experiments. � and �denote significant differences of values of AKT compared to untreated control. ^ and� denote significant differences of values of pAKT compared to untreated control.⁄p < 0.05 compared to untreated BPH-1 (CTRL). �p < 0.05 with respect to untreatedDU-145. ^p < 0.05 compared to untreated BPH-1 (CTRL). �p < 0.05 with respect tountreated DU-145.

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Fig. 8. Effects of TGF-b3 on PTEN expression in BPH-1, LNCaP and DU-145determined by Western blot. Experimental cells were treated with 10 and 50 ng/ml for 24 h. Data show the relative expression (mean + SEM) of calculated asarbitrary densitometric units (A.D.U.) collected from three independent experi-ments. ⁄p < 0.05 compared to untreated BPH-1 (CTRL). �p < 0.05 with respect tountreated DU-145.

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Bcl-2 25 KDa

BPH-1 LNCaP DU-145

Fig. 9. Effects of TGF-b3 on Bax/Bcl-2 expression in BPH-1, LNCaP and DU-145determined by Western blot. Experimental cells were treated with 10 and 50 ng/mlfor 24 h. Data show the relative expression (mean + SEM) of calculated as arbitrarydensitometric units (A.D.U.) collected from three independent experiments. � and ^denote significant differences of values of Bcl-2 compared to untreated control. �denote significant differences of values of Bax compared to untreated control.⁄p < 0.05, compared to untreated BPH-1 (CTRL). ^p < 0.05 compared to untreatedDU-145. �p < 0.05 with respect to untreated DU-145.

S. Caggia et al. / Cytokine 56 (2011) 403–410 407

pAKT in DU-145 after 24 h of TGF-b3 treatment. In Fig. 8 the resultsof expression of PTEN have been reported demonstrating that theeffects by TGF-b3 on this pro-apoptotic protein had a oppositebehaviour of PI3K/AKT/pAKT.

It has been demonstrated in vitro that nitric oxide (NO) can trig-ger apoptosis in mesangial cells and tubular cells, as well as other

cell types by mechanisms involving the up-regulation of p53, Baxand ceramide generation [32]. It has been suggested that long-lasting production of NO acts as a pro-apoptotic modulator by acti-vating the caspase family of proteases through the release of mito-chondrial cytochrome c into the cytosol, upregulation of p53expression, activation of c-Jun NH2-terminal kinase-stress-acti-vated protein kinase (JNK/SAPK), and altering the expression ofapoptosis-associated proteins including the Bcl-2 family of pro-teins [33]. Therefore, we determined the effect of TGF-b3 on Bax,

408 S. Caggia et al. / Cytokine 56 (2011) 403–410

and Bcl-2. The Bax/Bcl-2 ratio is a relevant relationship since it isgenerally recognized that maintenance of an appropriate Bax/Bcl-2 balance in cells prevents apoptosis [34]. Fig. 9 shows that com-pared to untreated respective controls the cellular level of Baxwas unchanged after 24 h of TGF-b3 treatment in BPH-1 andLNCaP, but it increased about 40% and further increased to 70%in TGF-b3-treated DU-145, respectively. Bcl-2 did not exhibit mod-ification in LNCaP by TGF-b3 at the two used concentrations, itshowed dose-dependent decrease in BPH-1 and increase in DU-145. Thus, the TGF-b3 treatment on BPH-1 and DU-145 causedthe relative Bax/Bcl-2 ratio to change from the normalized pre-treatment ratio of 1/1 to about 1.5/1 and 2/1, respectively.

4. Discussion

Work over recent years has shed light on the molecular mech-anisms underlying cancer. From the study of the pathways thatgovern tumour genesis and progression, a numerous amount of no-vel putative therapeutic targets have emerged, among them theTGF-b pathway. The purpose of this study was to explore howTGF-b3 might affect prostate tumour progression and to determinea potential role for TGF-b3 in the expression of YY1 and p53. Theresults of this research showed that cancer androgen-dependentLNCaP, cancer androgen-independent DU-145, and benign hyper-plasia BPH-1 cells answer in different manner to the treatmentwith 10 and 50 ng/ml of TGF-b3 for 24 h. TGF-b3 differently anddose-dependently influenced cell viability, YY1, p53, PI3K, Akt,pAkt, PTEN, Bax, Bcl-2 and iNOS expression, and NO productionof three cell lines. TGF-b3 demonstrated to be a potent anti-prolif-erative or pro-proliferative factor depending on the cell type, and itcan act as an inducer of apoptosis.

To date, up to 33 TGF-b-related genes have been identified inmammalian genomes as the result of genome sequencing project[29]. It utilizes a multitude of intracellular signalling pathways.The type III TGFb receptor (TGFbR3) has recently surfaced as a tu-mour suppressor within the prostate [35,36]. In a study on differ-ential gene expression of transforming growth factors alpha andbeta, epidermal growth factor, keratinocyte growth factor, andtheir receptors in foetal and adult human prostatic tissues and can-cer cell lines, Authors demonstrated that human BPH-1 cell linesand human prostate cancer cell lines (LNCaP, DU-145, PC-3) ex-press mRNA transcripts for TGF-b3 [37]. On stimulation from li-gand transfer, TGF–bR2 binds and phosphorylates the type Ireceptor (TGF–bR1), which in turn phosphorylates either theSmad2 or Smad3 transcription factor. Phosphorylation of Smad2/3 promotes binding to Smad4. The whole complex is then translo-cated into the nucleus where it regulates the transcription of myr-iad genes related to growth inhibition, production of theextracellular matrix, and apoptosis, among others [38]. Addition-ally, TGF-b binding to its receptors activates many non-canonicalsignalling pathways including PI3K, MAP kinase, and small GTPasepathways [39]. Several findings suggest a role in TGF-b signalling,which can rapidly activate PI3K, as indicated by the phosphoryla-tion of its downstream effector of Akt [29]. PI3K have been linkedto an extraordinarily diverse group of cellular functions, includingcell growth, proliferation, differentiation, motility, survival andintracellular trafficking. Many of these functions relate to the abil-ity of class I PI3K to activate protein kinase B (PKB, Akt) as in thePI3K/AKT/mTOR pathway. Akt/PKB is a serine/threonine protein ki-nase that plays a key role in multiple cellular processes such as glu-cose metabolism, cell proliferation, apoptosis, transcription andcell migration. The PI3K/mTOR pathway can be activated by over-production of growth factors or chemokines, loss of INPP4B orPTEN expression, or by mutations in growth factor receptors, Ras,PTEN, or PI3K itself. Activation of this pathway contributes to cell

growth, cell cycle entry, cell survival, and cell motility, all impor-tant aspects of tumorigenesis. A major role for PI3K pathway acti-vation in human tumours has been more recently establishedfollowing both the positional cloning of the PTEN tumour suppres-sor gene, and the discovery that the PTEN protein product was a li-pid phosphatase that antagonizes PI3K function and consequentlyinhibits downstream signalling through Akt [40]. Thus, TGF-b3 ap-pears to have a greater role in cancer development and progres-sion, and it is more than just an accessory protein.

YY1 is a multifunctional protein which can act as a transcrip-tional repressor or activator through combinatorial interactionwith other transcription factors, coactivators, corepressors andchromatin-remodelling complex. High levels of YY1 inhibit theexpression of p53 target genes after DNA damage [41]. Inhibitionof YY1 expression has been shown to modulate the cellular sensi-tivity to p53-mediated apoptosis in response to DNA damage [42].The numerous evidences that YY1 overexpression may promotep53 degradation or inhibit its transcriptional activity may give fur-ther insight into YY1’s role in cancer development.

The p53 tumour suppressor protein, also nicknamed the‘‘guardian of the genome’’, is a transcription factor that protectscells against a range of physiological stresses, such as oncogeneactivation, radiation, mitotic stress, ribosomal stress and chemicalinsults. These events lead to signals that are relayed to p53 whichgets activated, and through a complex network of interactions getslocated to the nucleus. There it turns on a program of transcriptionor repression of myriad genes that induce growth arrest, repair,apoptosis, senescence or altered metabolism; apoptosis is alsothought to be induced by direct interactions of cytoplasmic p53with mitochondria associated proteins. Due to its role as such acentral hub, it is no surprise that cells that lack p53 are prone totumours and these tumours are characterized by significant genet-ic abnormalities. In unstressed mammalian cells, p53 has a shorthalf-life and is normally maintained at low levels by continuousubiquitylation catalyzed by Mdm2 [43], COP1 (constitutively pho-tomorphogenic 1) [44], and Pirh2 (p53-induced protein with aRING-H2 domain) [45], and subsequent degradation by 26S protea-some. In this study, we showed the role of TGF-b3 in the modula-tion of the expression of YY1 and p53 on human benign prostatehyperplasia BPH-1, human prostate cancer androgen-non respon-sive DU-145 and androgen-responsive LNCaP cells. The resultsdemonstrated that TGF-b3 produces YY1 increase and p53 de-crease in BPH-1, no modification of YY1 and p53 in LNCaP, andYY1 decrease and p53 increase in DU-145. Some Authors haveshown that YY1 inhibits p300-mediated acetylation and henceblocks the stabilisation of p53 [41,24,46]. Additionally, YY1 stimu-lates ubiquitination of p53 and promotes its degradation. Conse-quently, They have demonstrated that ablation of endogenousYY1 correlates with increased p53 protein levels which is in goodagreement with the studies shown here (Figs. 2 and 3). We suggestthat, in DU-145 cells, TGF-b3 induces decrease of YY1 expression,and the mechanisms of acetylation and inhibited ubiquitinationlead to stabilised high p53 levels. Sui et al. [24] have indicated thatthe loss of YY1 expression in chicken cells induces an increase inthe sub-Go/G1 population, increased caspase activity and subse-quent apoptotic cell death.

The phosphatidylinositide 3-kinase (PI3K)/AKT-signalling path-way has been demonstrated to be a major survival signal in cancercells. The role of PI3K/Akt pathway in tumorigenesis has beenextensively investigated and altered expression or mutations ofmany components of this pathway have been implicated in humancancer [47]. Phosphorylated and activate Akt leads to inhibition ofapoptosis by inactivating several proapoptotic proteins, includingBad, Bax, and caspase 9, and also by inducing the expression ofantiapoptotic proteins such as Bcl-2, Bclxl, Flip, cIap2, Xiap, andsurvivins [48].

S. Caggia et al. / Cytokine 56 (2011) 403–410 409

Bcl-2 is the prominent member of a family of proteins that areresponsible for dysregulation of apoptosis and prevention of deathin cancer cells [34]. Antiapoptotic bcl-2 family members, includingbcl-xL, and proapoptotic proteins, such as Bad and Bax, interplaywith each other to control the pathways leading to the release ofcytochrome c from the mitochondrial membrane, the activationof caspase cascade and, finally, to the execution of apoptosis [34].Bcl-2 overexpression and/or activation has also been correlatedwith resistance to chemotherapy, to radiotherapy and to develop-ment of hormone-resistant tumours [49–51]. Moreover, it has beensuggested that Bcl-2 overexpression results in the up-regulation ofVEGF expression with increased neoangiogenesis in human cancerxenografts [52]. Therefore, Bcl-2 appears to be a relevant target forcancer therapy.

Current data show that hyper-physiological and physiologicallevels of p53 exert different effects on cellular redox status eitherthrough directly regulating the expressions of pro-oxidant andantioxidant genes or through modulating the cellular metabolicpathways [53]. Nitric oxide (NO) is an important bioactive mole-cule, which exerts diverse and sometimes opposing biological ef-fects. On the one hand, NO can mediate many beneficialphysiological processes such as protective cell killing by macro-phages, vasodilatation and various sorts of neurotransmission. Onthe other hand, elevated concentrations of NO can be detrimentalto cells because of the induction of inactivating protein modifica-tions, lipid oxidation, and DNA strand breaks [54]. Activation ofp53 by NO has been observed in many cell types. NO-inducedp53 contributes to various cell type specific biological effects ofNO, such as induction of apoptosis and inhibition of proliferation[55,56]. Most significantly, the crosstalk between NO and p53 islikely to play an important role in tumour suppression and in car-cinogenesis. Induction of p53 by NO is preceded by a rapid de-crease in Mdm2 protein, which may enable the initial rise in p53levels early after exposure to NO [54,57,58]. However, the molecu-lar mechanisms underlying the induction of p53 by NO have notbeen fully elucidated. NO can sensitize cells to p53-dependentapoptosis. The apoptotic effects of NO have already been shownto rely on p53 [55]. We show that, in DU-145 cancer cells, TGF-b3 by NO can cooperate with other p53-activating signals, suchas PI3K/Akt/PTEN, to maximize the contribution of p53 to cell kill-ing. This may be of particular relevance and such strategy may fur-ther benefit from the fact that the effect of TGF-b3 producing NOon p53 function and apoptotic activity is cell type-specific. Thismay provide an opportunity for selective killing of tumour cellsin an in vivo context by TGF-b3. NO is capable of stabilizing a trans-criptionally active p53 protein, likely through multiple mecha-nisms such as posttranslational modification, protein–proteininteractions, and of inhibition of degradation [33], in response toDNA damage, hypoxia, or oncogene activation. The ability of NOto promote a functional p53 response has been confirmed in mur-ine and human systems [59,60]. p53 in turn downregulates iNOSexpression through inhibition of the iNOS promoter [55], whereasp53 accumulation is attenuated by blocking NO formation [61,62].Our results confirm p53 stabilization in association with a celltype-specific differential expression of iNOS products by DU-145.

The results of this study demonstrated that TGF-b3: (i) in hu-man benign prostate hyperplasia BPH-1 cells, increases YY1 anddecreases p53; hypo-physiological levels of p53 increase expres-sion of PI3K, Akt, pAkt, and Bcl-2, decrease PTEN, and Bax, notmodify iNOS and NO; (ii) in androgen-responsive LNCaP cells, notalters YY1 and p53; physiological levels of p53 maintain cellularredox status; (iii) in human prostate cancer androgen-non respon-sive DU-14 cells, decreases YY1 and increases p53; hyper-physio-logical levels of p53 decrease expression of PI3K, Akt, pAkt, andBcl-2, increase PTEN, and Bax, activating pro-oxidant genes to pro-duce iNOS and NO. Since YY1 negatively regulates the expression

of Fas, probably the inhibition of YY1 by nitric oxide could upreg-ulate Fas and sensitize the DU-145 cells to Fas-induced apoptosis[63]. This induction of iNOS and NO is consistent with the kineticsof phosphorylation and nuclear translocation of Smad2 after TGF-binding to the signalling receptor complex.

Although more research is required to fully exploit the thera-peutic potential of TGF-b3 in prostate cancer, this study suggeststhat it might be an important target in the timely prostate cancer.This study records the profound differences in the responses of theBPH-1, LNCaP, and DU-145, cell lines to stimulation by TGF-b3.

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