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This article was downloaded by: [University of Glasgow]On: 08 October 2013, At: 05:53Publisher: RoutledgeInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,37-41 Mortimer Street, London W1T 3JH, UK
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Influence of Lycopene on Cell Viability, Cell Cycle,and Apoptosis of Human Prostate Cancer and BenignHyperplastic CellsNathalia da Costa Pereira Soares a , Anderson Junger Teodoro b , Felipe Leite Oliveira c ,Carlos Antonio do Nascimento Santos d , Christina Maeda Takiya e , Oswaldo Saback Junior f ,Mario Bianco f , Antonio Palumbo Junior g , Luiz Eurico Nasciutti g , Luciana Bueno Ferreira h
, Etel Rodrigues Pereira Gimba i j & Radovan Borojevic ka Programa de Pesquisa em Ciência de Alimentos, Instituto de Química, Universidade Federaldo Rio de Janeiro , Rio de Janeiro , Brazilb Laboratório de Bioquímica Nutricional, Programa de Alimentos e Nutrição, UniversidadeFederal do Estado do Rio de Janeiro , Rio de Janeiro , Brazilc Laboratório de Proliferação e Diferenciação Celular, Instituto de Ciências Biomédicas,Universidade Federal do Rio de Janeiro , Rio de Janeiro , Brazild Instituto Nacional de Metrologia, Qualidade e Tecnologia , Rio de Janeiro , Brazile Laboratório de Patologia Celular, Instituto de Ciências Biomédicas, Universidade Federal doRio de Janeiro , Rio de Janeiro , Brazilf Departamento de Cirurgia , Hospital Universitário Clementino Fraga Filho, UniversidadeFederal do Rio de Janeiro , Rio de Janeiro , Brazilg Programa de Pesquisa em Biologia Celular e do Desenvolvimento, Instituto de CiênciasBiomédicas, Universidade Federal do Rio de Janeiro , Rio de Janeiro , Brazilh Instituto Nacional do Câncer, Programa de Carcinogênese Molecular , Rio de Janeiro ,Brazili Universidade Federal Fluminense, Instituto de Humanidades e Saúde, DepartamentoInterdisciplinar , Rio das Ostras , Brazilj Instituto Nacional de Câncer, Programa de Carcinogênese Molecular , Rio de Janeiro , Brazilk Excellion Biomedical Services, Petrópolis , Rio de Janeiro , BrazilPublished online: 20 Sep 2013.
To cite this article: Nathalia da Costa Pereira Soares , Anderson Junger Teodoro , Felipe Leite Oliveira , Carlos Antoniodo Nascimento Santos , Christina Maeda Takiya , Oswaldo Saback Junior , Mario Bianco , Antonio Palumbo Junior , LuizEurico Nasciutti , Luciana Bueno Ferreira , Etel Rodrigues Pereira Gimba & Radovan Borojevic , Nutrition and Cancer (2013):Influence of Lycopene on Cell Viability, Cell Cycle, and Apoptosis of Human Prostate Cancer and Benign Hyperplastic Cells,Nutrition and Cancer, DOI: 10.1080/01635581.2013.812225
To link to this article: http://dx.doi.org/10.1080/01635581.2013.812225
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Nutrition and Cancer, 1–10, 2013Copyright C© Taylor & Francis Group, LLCISSN: 0163-5581 print / 1532-7914 onlineDOI: 10.1080/01635581.2013.812225
Influence of Lycopene on Cell Viability, Cell Cycle,and Apoptosis of Human Prostate Cancer and BenignHyperplastic Cells
Nathalia da Costa Pereira SoaresPrograma de Pesquisa em Ciencia de Alimentos, Instituto de Quımica, Universidade Federal do Rio deJaneiro, Rio de Janeiro, Brazil
Anderson Junger TeodoroLaboratorio de Bioquımica Nutricional, Programa de Alimentos e Nutricao, Universidade Federal doEstado do Rio de Janeiro, Rio de Janeiro, Brazil
Felipe Leite OliveiraLaboratorio de Proliferacao e Diferenciacao Celular, Instituto de Ciencias Biomedicas, UniversidadeFederal do Rio de Janeiro, Rio de Janeiro, Brazil
Carlos Antonio do Nascimento SantosInstituto Nacional de Metrologia, Qualidade e Tecnologia, Rio de Janeiro, Brazil
Christina Maeda TakiyaLaboratorio de Patologia Celular, Instituto de Ciencias Biomedicas, Universidade Federal do Rio deJaneiro, Rio de Janeiro, Brazil
Oswaldo Saback Junior and Mario BiancoDepartamento de Cirurgia, Hospital Universitario Clementino Fraga Filho, Universidade Federal doRio de Janeiro, Rio de Janeiro, Brazil
Antonio Palumbo Junior and Luiz Eurico NasciuttiPrograma de Pesquisa em Biologia Celular e do Desenvolvimento, Instituto de Ciencias Biomedicas,Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
Luciana Bueno FerreiraInstituto Nacional do Cancer, Programa de Carcinogenese Molecular, Rio de Janeiro, Brazil
Etel Rodrigues Pereira GimbaUniversidade Federal Fluminense, Instituto de Humanidades e Saude, Departamento Interdisciplinar,Rio das Ostras, Brazil; Instituto Nacional de Cancer, Programa de Carcinogenese Molecular,Rio de Janeiro, Brazil
Radovan BorojevicExcellion Biomedical Services, Petropolis, Rio de Janeiro, Brazil
Submitted 25 February 2013; accepted in final form 31 May 2013.Address correspondence to Nathalia da Costa Pereira Soares, Laboratorio de Proliferacao e Diferenciacao Celular, Instituto de Ciencias
Biomedicas, Av. Carlos Chagas, 373, Bloco F. 2◦ andar, Cidade Universitaria, Ilha do Fundao, Rio de Janeiro, Brasil 21941-902. Phone: +55 212562-6481. Fax: +55 21 2562-6483
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Prostate cancer is the most common malignancy in men andthe second leading cause of cancer-related mortality in men of theWestern world. Lycopene has received attention because of its ex-pcted potential to prevent cancer. In the present study, we evaluatedthe influence of lycopene on cell viability, cell cycle, and apoptosisof human prostate cancer cells and benign prostate hyperplasticcells. Using MTT assay, we observed a decrease of cell viabilityin all cancer cell lines after treatment with lycopene, which de-creased the percentage of cells in G0/G1 phase and increased inS and G2/M phases after 96 h of treatment in metastatic prostatecancer cell lineages. Flow citometry analysis of cell cycle revealedlycopene promoted cell cycle arrest in G0/G1 phase after 48 and96 h of treatment in a primary cancer cell line. Using real timePCR assay, lycopene also induced apoptosis in prostate cancercells with altered gene expression of Bax and Bcl-2. No effect wasobserved in benign prostate hyperplasia cells. These results sug-gest an effect of lycopene on activity of human prostate cancercells.
INTRODUCTIONProstate cancer is the most common malignancy in men,
and the second leading cause of cancer-related mortality in theworld. It is estimated that about 60.180 new cases of prostatecancer arise each year and about 10.000 result in death dueto complications of the disease (1, 2). Similar to other tissues,prostate is normally composed of cells that divide and replicatein an orderly and controlled manner, but it can also containcells with altered division patterns that give rise to benign ormalignant tumors (3, 4). In prostate, the benign excessive cellgrowth known as benign prostate hyperplasia (BPH) can coexistwith malignant prostate cancer (PCA), as well as with cells thatshow a normal growth pattern (5).
Currently, great attention has been given to strategies thatmay prevent cancer. Food compounds can act as anticarcino-genic, antioxidant, and antiinflammatory factors. They can alsoact against cancers that are induced or sustained by hormones,or they may have antiangiogenic activity (6). Among the sub-stances with potential chemopreventive activity, lycopene hasattracted interest because of its possible potential to preventPCA and cardiovascular diseases.
Plants are the main source of carotenoids in food, and up-take of carotenoid-rich compounds was inversely correlatedwith cancer in numerous epidemiological studies and in vitroassays (7–9). Multiple possibilities can explain the biologicaleffects promoted by carotenoids. Some are provitamin-A, be-ing converted to retinoids, participating thus in retinoic acidsignaling intracellular pathways. They may also modulate enzy-matic activities, act as proinflammatory and immunomodulatorymolecules, promote antioxidant activity, or modulate intercel-lular communications through controls of connexin-43 genes(10–12).
Although not considered to be an essential nutrient, lycopenecan bring many benefits to human health (13). The antioxidantfunction has been widely investigated and it is possibly associ-
ated with some of the effect of diets rich in lycopene. Uptakeof dietary lycopene increases the status of serum lycopene thatmay help to reduce the occurrence and progression of esoph-agus, stomach, prostate, and lung cancer (14). The evidenceof the role of lycopene in the prevention of chronic diseasescomes both from epidemiological studies (15, 16) and cell cul-ture studies using human cancer cell lines (17), as well as studiesin animals (18) and in human clinical trials (19), but data on invivo effects of lycopene are scarce.
Kim et al. (17) using different concentrations of lycopene inhuman PCA cell lines showed that it was able to considerablyreduce the cell cycle progression in all the studied periods.Although the exact mechanism by which lycopene reduces thegrowth of PCA cells is still not well understood, it is frequentlybelieved that this effect may be due to the protective action oflycopene against oxidative damage caused by reactive oxygenspecies in cells. Lycopene is indeed found in the human prostatetissue, suggesting the biological possibility of a direct effect ofthis carotenoid in prostate function and carcinogenesis (20).
In the present study we evaluated the influence of lycopene oncell viability, cell cycle, and apoptosis in 2 established metastaticPCA cell lines (PC-3 and DU-145), a primary culture of humanPCA cells and of BPH.
MATERIAL AND METHODS
ReagentsAll-trans lycopene was purchased from Sigma Chemical
Company (St. Louis, MO). Water-soluble lycopene (10%) wasprovided by Roche (Rio de Janeiro, Brazil). Dulbecco’s cellculture medium and bovine serum albumin were obtained fromSigma, and fetal bovine serum (FBS) from Laborclin (Camp-inas, Brazil). Tissue culture flasks were obtained from Nunc(Roskilde, Denmark). All the chemicals were of analyticalgrade.
Cell Culture and Treatment ProtocolAll the cells lines were obtained from the Rio de Janeiro
Cell Bank (Inmetro Tecnological Park, Xerem, Rio de Janeiro,Brazil), in which absence of contaminants and the identity ofcell lines were checked and controlled. Bone metastasis-derivedPCA human cells (PC-3), and brain metastasis-derived PCAhuman cells (DU-145) were plated in 25 cm2 tissue cultureflasks, 5.0 × 106 cells/flask, and maintained routinely in theDulbecco’s medium (DMEM) supplemented with 10% FBSand 2 g/L HEPES buffer, pH 7.4, under 5% CO2 atmosphere.Stock flasks were grown to 70% confluence and subculturedroutinely. Medium renewal was done 2–3 times weekly. For eachexperiment, all the cells were seeded at 104 cells/cm2 densityin 6- and 96-well plates for cell cycle and cell proliferationanalyses, respectively. After 24 h, medium was removed andcells were treated with increasing concentrations of lycopene(0.5; 1; 2.5; 5; 10 and 20 μM) dissolved in DMEM at 50◦C.
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The controls were included on each plate. The cells were thenincubated for 48 and 96 h with daily medium replacement (21).
Isolation and Characterization of Primary Cell CulturesTransurethral resection fragments of prostate tissues from 2
BPH surgeries were used to obtain the BPH cells. PCA cellswere obtained from fragments of prostate tissues obtained from2 cancer cases submitted to radical prostatectomy. Participantsprovided their written consent to participate in this study uponsignature on the Consent Term established. The procedures wereapproved by the Ethics Committee of the Clementino FragaFilho University Hospital, Federal University of Rio de Janeiro,Protocol-CAAE0029.0.197.000-05.
Tissues were cut in fragments of 1–3 mm3 that were grown in24-well plates containing DMEM supplemented with 10% FBSand 1 μL/mL penicillin (Sigma). The medium was changed ev-ery 2 days. Cells were trypsinized and transferred to 25 mm2
culture dishes. After six passages, a homogeneous cell popula-tion was obtained.
The cells were immunocytochemically characterized as fol-lows. Cells were washed twice with PBS and fixed with 4%paraformaldehyde-PBS (Sigma) for 10 min. After fixation, theywere washed with PBS and incubated in a 50 nM NH4Clfor 30 min. The nonspecific antibody binding was blockedwith PBS/BSA 5%, and the primary antibodies were incubatedovernight. We used antibodies against vimentin (Sigma) andα-smooth muscle actin (Sigma) for BPH. We used antibodiesagainst cytokeratin 5 (Cell Marque) and alpha-methylacyl CoAracemase (P504S-Cell Marque) for primary PCA cells. After in-cubation with primary antibodies, cells were washed with PBSand incubated for 2 additional hours with either goat antimouseAlexa 488 or goat antirabbit Alexa 488/546 secondary anti-bodies (Invitrogen). Cell nuclei were stained with DAPI (SantaCruz Biotechnology). Finally, cells were washed in distilledwater and mounted on histological slides with N-propylgallate(Sigma). Images were captured using a confocal microscopy(Olympus IX81) and a Hamamatsu OrcaR2 digital camera.
Cell Viability AssayCell viability was monitored by MTT assay (Am-
resco, Solon, OH). MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) is a pale yellow substrate thatis reduced by living cells to yield a dark blue formazan product.This requires active mitochondria, and even recently dead cellsdo not reduce significant amounts of MTT. Exponentially grow-ing cells were adjusted to 1.0 × 104/cm2 with DMEM, platedin 96-well plates (Corning, Tewksbury, MA) at 200 μL/welland incubated for 12 h according to the routine procedure. Thecells were then incubated with lycopene (0.5–20 μM) for 48 hand 96 h (6 wells for each sample). Each well was also incu-bated with MTT (20 μL/well; 5 g/L) for 4 h. The liquid wasremoved and 100 μL/well sodium dodecyl sulfate was added todissolve the solid residue. Finally, the absorbance was measuredusing a microplate reader (BIO-RAD 2550) at 570 nm. The cell
proliferation inhibition rate (CPIR) was calculated using the fol-lowing formula: CPIR = (1 – average A value of experimentalgroup/average A value of control group) × 100%.
Cell Cycle AssaysCell cycle analysis was done using the propidium iodide
assay. Briefly, cells were resuspended in 500 μL propidiumiodide solution (PBS, 0.1% Triton X-100, 0.1% RNAse and50 μg/mL propidium iodide; Sigma). Cell cycle analysis wasassessed by flow cytometry (FACScalibur, BD Bioscience, SanJose, CA), and after acquisition of 20,000 events the data wereanalyzed in Cell Quest software.
Apoptosis AssaysIn apoptotic cells, phosphatidylserine translocates from the
inner to the outer leaflet of the cell membrane, where annexinV conjugated to fluorescein isothiocyanate (FITC) can bind.Coupled with propidium iodide staining, disruption or increasedpermeability of the plasma membrane can also be determined.
Cells were resuspended in 400 μL binding buffer containing5 μL of annexin V FITC and 5 μL propidium iodide (Apopto-sis Detection Kit II, BDBiosciences) for 15 min at room tem-perature. Annexin V binding was evaluated by flow cytometry(FACScalibur, BD Biosciences), and after acquisition of 20,000events the data were analyzed in Cell Quest software.
Gene Expression AnalysisTotal RNA from the studied cells was extracted using RNeasy
Mini Kit (Qiagen) according to the manufacturer’s instructions.RNA yield and quality were determined by a spectrophotometerNanoDrop ND-1000 V3.2 (Nanodrop Technologies, Wilming-ton, DE). Equal amounts (1 μg) of RNA from cells were re-verse transcribed with cDNA Syntesis kit “Superscript II First-Strand Synthesis System for RT-PCR” (Invitrogen) and Oligo(dT) primer (Invitrogen). The cDNA was used as a templatefor subsequent real-time polymerase chain reaction (RT-PCR).Quantitative RT-PCR was done in a CFX96 Real Time System(BIORAD) C1000 Thermal Cycler using SYBR Green (AppliedBiosystems, Grand Island, NY) following the manufacturer’s in-structions. The expression level of CK18, Bax, and Bcl-2 mRNAwere all normalized with β-actin expression level. To evaluatethe quality of the RT-PCR products, melt curve analyses wereperformed after each assay. Relative expression was determinedusing the ��CT method with β-actin rRNA as the referencegenes.
Statistical AnalysisThe presented data are mean values ± standard error of 3
independent experiments done in duplicate (n = 6). Statisti-cal comparisons were carried out by analysis of variance andpost hoc Tukey’s test using Graph Pad Prism 5.0 and Statistical6.0 program. The differences were considered significant whenP < 0.05.
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FIG. 1. Characterization of benign prostate hyperplasia and primary prostate cancer cells. Phase-contrast micrograph of primary benign hyperplasia prostatecells (A and B) and prostate cancer cells (G and H). Immunostaining for vimentin (C and D) and α-smooth muscle actin (E and F) in benign prostate hyperplasiacells. In prostate cancer cells, immunostaining for CK5 (I and J) and racemase (K and L) (Color figure available online).
RESULTS
Characterization of CellsA homogeneous primary cell population was obtained from
BPH surgery resections. The primary cell culture was charac-terized at each passage and we observed a gradual decrease inthe number of rounded cells with epithelial characteristics andan increase in the number of elongated cells with fibroblasticaspect (Fig. 1A and 1B). Previous studies have shown that pri-mary prostate cell cultures were characterized by an increase inexpression of cytoskeleton proteins, such as vimentin (Fig. 1Cand 1D) and α-smooth muscle actin (Fig. 1E and 1F). After 6passages, we observed a cell population staining for vimentinand α-smooth muscle actin. The latter one was diffuse and faintin the small actively proliferating cells, but large bundles ofstress fibers were observed in differentiated well spread cells.These results indicated that a homogenous BPH cell populationwas isolated, characterized as prostate stroma cells.
Similar to BPH cells, a PCA homogeneous population cellwas obtained from primary PCA tissue (Fig. 1G and 1H). Thesecells were characterized by expression of cytokeratin 5 (CK5),a marker of basal proliferating cells in the prostate epithelium(Fig. 1I and 1J). They were also positive for alpha-methylacylCoA racemase (Fig. 1K and 1L), which is considered to be a use-
ful marker for neoplastic transformation in the prostate(22–23).Iwasa et al. (24) demonstrated presence of this racemase in about94% prostate adenocarcinomas.
Effect of Lycopene on Cell ViabilityAll cell lines had normal growth characteristics expected
under standard in vitro culture conditions. Previous studies re-ported that lycopene formulated as 10% water soluble granuleswas not toxic. Hereafter, all the results refer to lycopene withthis vehicle, already used in previous studies in vitro (25). Theplating of cancer cell lines was followed by 24 h recovery,and cells were subsequently incubated with 0.5, 1, 2.5, 5, 10,or 20 μM lycopene during 24, 48, 72, and 96 h. All the datashowed no effect of lycopene after 24 h incubation and theyare not further represented. We used the MTT assay to monitorthe cell viability. In DU-145 cell line, lycopene did not changecellular growth profile within 48 h of treatment (P > 0.05)(Fig. 2A). At 72 h lycopene promoted an inhibition of 20% at5 μM that increased to 25% at 10 μM and remained at thesame level at 20 μM, indicating potentially a saturation of thelycopene uptake or metabolism (Fig. 2B).
In PC-3 cells, lycopene showed inhibitory growth effectof viable cells when compared to the control group, after 48
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FIG. 2. Effect of lycopene on viability of DU-145 (A and B) and PC-3 (C and D) cells after exposure using MTT assays. Forty-eight hours (A and C) and 96 h(B and D). The experiment is expressed as mean ± standard error and differences significant between treated cells with lycopene (5–10 μM) were compared usingthe Tukey test (∗P < 0.05, ∗∗P < 0.01).
and 96 h of treatment with the highest lycopene concentra-tion only (20 μM), with an average reduction of 15% (Fig.2C and 2D). No increase was observed from 48 to 96 h oftreatment.
Lycopene decreased cell viability in PCA cells within 48 h,from the 1 μM lycopene concentration on (Fig. 3A). At 72 hincubation, the cell growth inhibition remained similar to 48 h(data not shown) (P > 0.05) but the inhibitory effect couldbe observed also for lower lycopene doses (0.5 μM) (data notshown). A potent inhibitory effect on PCA cell viability at 96 h oftreatment was observed, reaching 40% (Fig. 3B). No statisticaldifference was observed between the used doses indicating thatthe saturation of the lycopene activity was possibly reached indoses lower than 0.5 μM.
No change on cell viability was induced in BPH cells (Fig.3C and 3D) treated with lycopene at any time interval examinedor carotenoid concentration used.
Taken as a whole, these data indicate that lycopene’s effectwas cell-specific and time-dependent, and that lycopene can bea potent inhibitor of human PCA cells growth.
Effect of Lycopene on Cell Cycle ProgressionTo monitor the influence of lycopene on cell cycle, we treated
cells with lycopene (5–10 μM) for 48 and 96 h and quantifiedthe cells percentage in different cell cycle phases.
The DU-145 cell line showed an increase in cells retainedin the G0/G1 phase, followed by a decrease of cells in G2/M
phase when treated with the two concentrations of lycopene,after 48 h (Fig. 4A). After 96 h treatment, a reverse effect wasobserved, and lycopene induced an accumulation of cells in Sand G2/M phases followed reduction in percentage of cells inG0/G1 phase (Fig. 4B).
In PC-3 cells, lycopene modulated the cell cycle progressioninducing a reduction in the cells percentage in G0/G1 phase,after 48 h treatment. The effect was maintained after 96 h treat-ment, with an increase in accumulation of cells in G2/M phaseand reduction in G0/G1 phase (Fig. 4A and 4B).
In PCA cells, an increase of cells in G0/G1 phase and a de-crease in G2/M phase were observed for both times of treatmentwith lycopene (Fig. 5A and 5B).
In human benign prostate hyperplasic cells lycopene did notpromote any change of the cell cycle progression, regardless oftreatment time and/or dose used (Fig. 5A and 5B).
ApoptosisQuantification of apoptosis can be a useful measure of can-
cer cell kinetics (Fig. 6). Alterations of the balance betweenproliferation and apoptosis are associated with cancer. We havestudied cancer cells incubated with lycopene (5–10 μM) for 48and 96 h.
In DU-145 cell line we observed an up to fivefold increaseof apoptotic cells after 96 h exposure to lycopene at 10 μMconcentration. The effect of lycopene on apoptosis in PC-3 cellline was lower as compared to DU-145 cell line, with maximal
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FIG. 3. Effect of lycopene on viability prostate cancer (A and B) and benign prostate hyperplasia (C and D) cells after exposure using MTT assays. Forthy-eighthours (A, C) and 96 h (B, D). The experiment is expressed as mean ± error standard and differences significant between treated cells with lycopene (5–10 μM)were compared using the Tukey test (∗P < 0.05).
FIG. 4. Effect of lycopene on cell cycle progression in DU-145 and PC-3 cell lines after 48 h and 96 h exposure. The cell cycle phases and quantitative resultsare illustrated at A (48 h) and B (96 h) in accordance with the exposure time and carotenoid concentration. The experiment is expressed as mean ± error standardand differences significant between untreated cells (Control) and treated with lycopene (5–10 μM) were compared using the Tukey test (∗P < 0.05, ∗∗P < 0.01).
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FIG. 5. Effect of lycopene on cell cycle progression in benign prostate hyperplasia and prostate cancer cells after 48 h and 96 h exposure. The cell cycle phasesand quantitative results are illustrated at A (48 h) and B (96 h) in accordance with the exposure time and carotenoid concentration. The experiment is expressedas mean ± error standard and differences significant between untreated cells (Control) and treated with lycopene (5–10 μM) were compared using the Tukey test(∗P < 0.05, ∗∗P < 0.01).
increase 2.2-fold after 48 h at 10 μM concentration. These dataare in agreement with the results described above related toviability and cell cycle.
PCA and BPH cells showed that lycopene promoted apopto-sis in PCA cells with an average increase 1.35-fold after 48 htreatment and maximum increase 2.25-fold after 96 h, at thehighest lycopene concentration (10 μM). In BPH cells no sig-nificant difference was observed when compared to the untreatedgroup, results similar to those obtained in analysis of viabilityand cell cycle.
Lycopene Induce an Antitumorigenic GeneExpression Profile
To study putative molecular mechanisms by which lycopeneinterferes in prostate tumor progression, we investigated ex-pression profile of several related genes (Fig. 7). We have com-pared DU-145 cell line obtained from metastatic growth of PCA,which has shown a high response to lycopene, with a primaryculture (PCA) of an in situ PCA, expected to be less aggressive.In DU-145 cells, lycopene treatment promoted a significant up-regulation Bax and CK18 genes, whereas the expression of Bcl-2genes was downregulated (Fig. 7A).
In PCA cell, lycopene treatment promoted an upregulationof Bax and CK18 genes and a downregulation of Bcl-2 gene(Fig. 7B).
DISCUSSIONThe present study has given several sets of information that
may be relevant for understanding lycopene interaction withprostate cells. We have shown the specificity of lycopene effecton cells in the used experimental model, a consistent timingand dose response of cells to lycopene, as well as modificationsof cell cycle and apoptosis in PCA but not in hyperplasic butnoncancerous prostate stroma cells.
PCA is the second most common cancer in men and epidemi-ological evidence indicates that the consumption of tomatoes ortomato-derived alimentary products and the risk of PCA areinversely correlated, but the same was not observed for supple-mentation with lycopene alone (26).
The cells obtained to the primary culture of BPH were theconnective tissue stroma cells. The BPH cells did not respondto lycopene in any of the studied experimental settings. Thisresult was expected, because the BPH cells are not hormone-dependent, these data are consistent with the proposal that ly-copene interacts with steroid hormone signaling (27). We mayconclude that lycopene activity is cell type dependent. Thesame results confirm that the lycopene, in concentrations usedthroughout the study, is not toxic and does not interfere with thebasic metabolic activities of the cells.
The presently observed timing and dose-response of all thestudied cells to lycopene are somewhat surprising and indi-cate the required further studies of lycopene interactions with
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FIG. 6. Effect of lycopene on programmed cell death after 48 h and 96 h exposure. The flow cytometric analyzes are shown according to the exposure time andcarotenoid concentration. The quantitative results of lycopene on cell lines are shown after 48 and 96 h. The experiment is expressed as mean ± error standardand differences significant between untreated cells (Control) and treated with lycopene (5–10 μM) were compared using the Tukey test (∗P < 0.05, ∗∗P < 0.01,∗∗∗P < 0.001).
FIG. 7. Profile of gene expression in human prostate cancer cells. Quantitative analysis of real-time PCR in different genes associated with cancer progression,after 96 h incubation with lycopene. Transcription levels RNA β-actin gene were used as internal control. The experiment is expressed as mean ± error standardand differences significant between treated cells with lycopene (5–10 μM) were compared using Tukey test (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).
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INFLUENCE OF LYCOPENE ON PROSTATE CANCER 9
cells. In none of the assayed experimental conditions, lycopeneshowed a significant effect on cells at 24 h. Subsequently, inmore sensitive cells such as PCA, the effect was observed from48 h on, but in most cases the full effect was reached only afterlonger periods of incubation.
In a previous study of lycopene uptake in hepatic perisinu-soidal stellate cells, the cell type specialized in retinoid uptakeand storage, we have shown the 2-phase pattern of lycopeneuptake (25). The first one is basal and not modified from 12 to48 h of incubation. It increases steadily from 72 h on, but theincrease is not dependent of the extracellular lycopene level, in-dicating that it can be potentially saturated. The same pattern ofresponse was now observed in PCA cells, in which the increaseof extracellular lycopene generally did not lead to the dose-dependent response. We have no evidence of lycopene storagein prostate cells, and the kinetics of uptake and metabolism oflycopene are at present under study. The 48 h delay in effi-cient lycopene uptake and activity is compatible with inductionof protein synthesis, either of carriers or of enzymes that maymodify membrane properties and/or lycopene downprocessing.Hepatic stellate cells are specialized in storage of retinol, whichinteracts with both plasmatic and intracellular specific carriers,who are induced upon exposition to retinol and who control itsuptake and metabolism (28). In conclusion, it may be proposedthat long-term and continuous, but not necessarily high contentof lycopene in the cell environment may be the most relevantfor its protection in PCA.
The cell proliferation, cell cycle, and apoptosis are coupled inkinetics of normal and neoplastic cell growth. The effect of ly-copene was different in the three cell types studied. The highestprotective effect was observed on PCA cells. These are primarycancer cells with typical epithelial characteristic, correspondingto the basal proliferative cells of the prostate epithelium. Assuch, they are expected to be hormone-dependent and sensitiveto the local environment. They showed relatively early and highproliferation inhibition in presence of low quantity of lycopene(2.5 μM), which is in the range of lycopene concentration inblood of healthy persons with diet rich in fruits and vegetablesthat are sources of carotenoids, reported to be in the range of3.8 μM (29). The inhibitory effect was already saturated at 96 hwith this low lycopene content and it did not increase with muchhigher extracellular lycopene, indicating again that lower butsustained concentration may reach the maximal inhibition level.
The cell cycle modification in PCA cells by lycopene in-volved a marginal increase in G0/G1 and the correspondingdecrease in G2/M. The cell arrest in G0/G1 can be long; it isreversible and it is of lesser importance for a cell population insitu as compared to the arrest in G2/M, which leads to apop-tosis when cells cannot recover and proceed to cell division.However, PCA cells showed a significant increase in apoptosis,suggesting that another mechanism may be involved. Indeed,the equilibrium of Bcl2/Bax expression was highly modified inPCA cells exposed to lycopene, and this cue may be relevantfor understanding of lycopene effect of on PCA cells.
The 2 studied PCA cell lines, DU-145 and PC-3, are derivedfrom distant metastases of PCA. As such, they have passedthrough the epithelial-mesenchymal transition and are expectedto be different both from primary PCA and between them, be-cause each established cancer lineage passes through extensiveselection both in vivo and in subsequent culture in vitro. TheDU-145 showed a higher inhibition of proliferation in elevatedlevels of lycopene as compared to the PC-3 cell lineage. It had asignificant increase in G2/M arrest, which may lead to apoptosis,and this was indeed observed in the apoptotic cell quantifica-tion. A lesser effect was observed on the Bcl2/Bax expression,and we understand that different from PCA cells, the alterationof the cell cycle may be the major cause of lycopene inhibitoryeffect on this cell lineage.
Cell-cycle deregulation is a fundamental aspect in cancer de-velopment. Previous studies reported that lycopene induced aG1/S cell cycle arrest, which is corroborated by the downregu-lation of cyclins, including cyclin E and cyclin D1 and/or by theupregulation of cyclin A and p27 (11, 30, 31). Whilst arrest inG0/G1 can be reverted, and cells can proceed with proliferationafter interruption of the treatment, G2/M arrest leads potentiallyto apoptosis.
The progressive malignant transformation of prostate epithe-lial cells depends upon transition from basal to luminal phe-notypes. Basal cells are proliferative and sustain continuouslyrenewal of the differentiated luminal secretory cells that are of ashort life span and reduced proliferative capacity (32, 33). Thesetwo epithelial cell populations differ in their morphology, pat-tern of cytokeratin expression and of hormonal responsiveness.Luminal cells are characterized by the expression of CK8/CK18,and are dependent upon steroid hormones. Basal cells expressCK5 and are androgen-independent (34). Stromal cells are im-portant for proliferation of prostate epithelial cells, because theyprovide type required supplementary growth factors and adhe-sive ligands (35, 36).
Our study showed that lycopene positively regulates CK18expression in all the cells analyzed, indicating induction oftheir engagement in the terminal differentiation. Comparisonof CK18 expression with different clinical risk factors showeda highly significant correlation among the size, differentiationgrade, and mitotic index of the primary tumor. These param-eters are related to a proliferation rate of the primary tumor,suggesting a possible relationship between downregulation ofCK18 expression and increased proliferative activity, which isapparently reverted by lycopene (37).
We demonstrated that lycopene inhibited cell proliferation,arrested cell cycle in different phases, and increased apoptosisin human PCA cells. Taken together, the present in vitro studysupport the theory that lycopene may have a protective effecton PCA, because it seems to be more efficient in terms of in-hibition of the cells of early stages of abnormal growth withinthe prostate epithelium. In some cases, it can decrease the latePCA events through alteration of the cell cycle, but the low re-sponse of the PC-3 cell line makes the proposal less strong. The
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protective effect in early stages is potentially more plausiblethan the curative effect of lycopene in late PCA.
ACKNOWLEDGMENTSThis research received financial support from CAPES
(Coordenacao de Aperfeicoamento de Pessoal de Nıvel Supe-rior), CNPq (Conselho Nacional de Desenvolvimento Cientıficoe Tecnologico) and FAPERJ (Fundacao de Amparo a Pesquisado Estado do Rio de Janeiro).
REFERENCES1. Syed DN, Khan N, Afaq F, and Mukhtar H: Chemoprevention of prostate
cancer through dietary agents: progress and promise. Cancer EpidemiolBiomarkers Prev 16, 2193–2204, 2007.
2. Brasil Ministerio da Saude, Secretaria de Assistencia a Saude: Estima-tiva de novos casos de cancer de prostata 2012. Instituto Nacional doCancer, 2012. Retrieved from http://www2.inca.gov.br/wps/wcm/connect/tiposdecancer/site/home/prostata
3. Correa NAB, Costa GFM, Massambani EM, Matumoto FH, and PaulaMMM: Diagnostico precoce de carcinoma de prostata: antıgeno prostaticoespecıfico (PSA), um marcador quase ideal. Rev Bras Anal Clin 35, 63–64,2003.
4. Dini LI and Koff WJ: Perfil do cancer de prostata no hospital de clınicas dePorto Alegre. Rev Assoc Med Bras 52, 28–31, 2006.
5. Srougi M and Simon SD (Eds.): Cancer urologico, 2nd ed. Ed. Platina, SaoPaulo, Brazil, 1996.
6. Upadhyaya KR, Radha KS, and Madhyastha HK: Cell cycle regulation andinduction of apoptosis by beta-carotene in U937 and HL-60 leukemia cells.J Biochem Mol Biol 40, 1009–1015, 2007.
7. Rao AV and Rao LG: Carotenoids and human health. Pharmacol Res 55,207–216, 2007.
8. Ren D, Peng G, Huang H, Wang H, and Zhang S: Effect of rhodoxanthinfrom Potamogetoncrispus L. on cell apoptosis in Hela cells. Toxicology InVitro 20, 1411–1418, 2007.
9. Slattery ML, Benson J, Curtin K, Ma K, Schaeffer D, et al.: Carotenoidsand colon cancer. Am J Clin Nutr 71, 575–582, 2000.
10. Paiva SAR and Russell RM: Beta-carotene and other carotenoids as antiox-idants. J Am Coll Nutr 18, 426–433, 1999.
11. Palozza P, Serini S, Maggiano N, Angelini M, Boninsegna A, et al.: In-duction of cell cycle arrest and apoptosis in human colon adenocarcinomacell lines by beta-carotene through down-regulation of cyclin A and bcl-2family proteins. Carcinogenesis 23, 11–18, 2002.
12. Rodriguez-Amaya DB. A Guide to Carotenoid Analysis in Foods. Interna-tional Life Sciences Institute Press, Washington, DC, 2001.
13. Levy J and Sharoni Y: The Functions of Tomato Lycopene and Its Role inHuman Health. J American Botanical Council 62, 49–56, 2004.
14. Clareto SS: Estudo da Concentracao de Licopeno da Polpa de GoiabaUtilizando o Processo de Microfiltracao. Brasil: Tese de Doutorado emTecnologia de Alimentos UNICAMP, 2007.
15. LaVecchia C: Mediterranean epidemiological evidence on tomatoes and theprevention of digestive tract cancers. Proc Soc Exp Bio Med 218, 125–128,1997.
16. Giovannucci E, Liu Y, Stampfer MJ, and Willett WC: A prospective studyof tomato products, lycopene and prostate cancer risk. J Natl Cancer Inst94, 391–398, 2002.
17. Kim L, Rao AV, and Rao LG: Effects of Lycopene on prostate LNCaPcancer cells in culture. J Medicinal Food 5,181–187, 2002.
18. Rao AV: Lycopene and human health: summary and future directions. In:Tomatoes, Lycopene and Human Health. Ardersier, Scotland: CaledonianScience Press, 2006, pp. 223–228.
19. Bowen P, Chen L, and Stacewicz-Sapuntzakis M: Tomato sauce supple-mentation and prostate cancer: Lycopene accumulation and modulation ofbiomarkers of carcinogenesis. Exp Biol Med 10, 886–893, 2002.
20. Shami NJIE and Moreira EAM: Licopeno como agente antioxidante. Re-vista de Nutricao 17, 227–236, 2004.
21. Levy J, Bosin E, Feldman B, Giat Y, Miinster A, et al.: Lycopene is amore potent inhibitor of human cancer cell proliferation than either alpha-carotene or beta-carotene. Nutrition and Cancer 24, 257–266, 1995.
22. Jiang Z, Wu CL, Woda BA, Dresser K, Xu J, et al.: P504S/alpha-methylacyl-CoA racemase: a useful marker for diagnosis of small foci of prostaticcarcinoma on needle biopsy. Am J Surg Pathol 26, 1169–1174, 2002.
23. Molinie V, Herve JM, Lebret T, Lugagne-Delpon PM, Saporta F, et al.:Value of the antibody cocktail anti p63 + anti p504s for the diagnosis ofprostatic cancer. Ann Path 24, 6–16, 2004.
24. Iwasa Y, Mizokami A, Miwa S, Koshida K, and Namiki M: Establishmentand characterization of androgen-independent human prostate cancer celllines, LN-REC4 and LNCaP-SF, from LNCaP. Int J Urol 14, 233–239,2007.
25. Teodoro AJ, Perrone D, Martucci RB, and Borojevic R: Lycopene isomeri-sation and storage in an in vitro model of murine hepatic stellate cells. EurJ Nutr 48, 261–268, 2009.
26. Ellinger S, Ellinger J, and Stehle P: Tomatoes, tomato products and lycopenein the prevention and treatment of prostate cancer: do we have the evidencefrom intervention studies? Curr Opin Clin Nutr Metab Care 9, 722–727,2006.
27. Liu X, Allen JD, Arnold JT, and Blackman MR: Lycopene inhibits IGF-1signal transduction and growth in normal prostate epithelial cells by de-creasing DHT-modulated IGF-1 production in co-cultured reactive stromalcells. Carcinogenesis 29, 816–823, 2008.
28. Ambrosio DN, Clugston RD, and Blaner WS: Vitamin A metabolism: anupdate. Nutrients 3, 63–103, 2011.
29. Yang Z, Zhang Z, Penniston KL, Binkley N, and Tanumihardjo SA: Serumcarotenoid concentrations in postmenopausal women from the United Stateswith and without osteoporosis. Int J Vitam Nutr Res 78, 105–111, 2008.
30. Nahum A, Hirsch K, Danilenko M, Watts CK, Prall OW, et al.: Lycopeneinhibition of cell cycle progression in breast and endometrial cancer cellsis associated with reduction in cyclin D levels and retention of p27(Kip1)in the cyclin E-cdk2 complexes. Oncogene 20, 3428–3436, 2001.
31. Ivanov NI, Cowell SP, Brown P, Rennie PS, Guns ES, et al.: Lycopenedifferentially induces quiescence and apoptosis in androgen-responsive and-independent prostate cancer cell lines. Clin Nutr 26, 252–263, 2007.
32. Coffey RN, Watson RW, Hegarty PK, Watson CL, Wolohan L, et al.:Priming prostate carcinoma cells for increased apoptosis is associated withupregulation of the caspases. Cancer 92, 2297–2308, 2001.
33. Peehl DM: Primary cell cultures as models of prostate cancer development.Endocrine-Related Cancer 12, 19–47, 2005.
34. Peehl DM: Are primary cultures realistic models of prostate cancer? J CellBiochem 91, 185–195, 2004.
35. Barclay WW, Woodruff RD, Hall MC, and Cramer SD: A system forstudying epithelial-stromal interactions reveals distinct inductive abilitiesof stromal cells from benign prostatic hyperplasia and prostate cancer.Endocrinology 146, 13–18, 2005.
36. Cunha GR, Hayward SW, Wang YZ, and Ricke WA: Role of the stromalmicroenvironment in carcinogenesis of the prostate. Int J Cancer 107, 1–10,2003.
37. Kramer G, Erdal H, Mertens HJ, Nap M, Mauermann J, et al.: Differen-tiation between cell deathmodes using measurements of different solubleforms of extracellular cytokeratin 18. Cancer Res 64, 1751–1756, 2004.
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