potential chemopreventive activities of a polyphenol rich ......potential chemopreventive activities...

Potential chemopreventive activities of apolyphenol rich purified extract from olive millwastewater on colon cancer cells

Barbara Bassani a,1, Teresa Rossi b,1, Daniela De Stefano a,Daniele Pizzichini c, Paola Corradino a, Nicoletta Macrì a,Douglas M. Noonan a,d, Adriana Albini a,*,1, Antonino Bruno a,1

a Scientific and Technology Pole, IRCCS MultiMedica, Via G. Fantoli 16/15, Milan 20138, Italyb Laboratory of Translational Research, IRCCS Arcispedale Santa Maria Nuova, Viale Risorgimento, 80, ReggioEmilia 42123, Italyc ENEA, Casaccia Research Center, Via Anguillarese, 301, Rome 00123, Italyd Department of Biotechnologies and Life Sciences, University of Insubria, Via O. Rossi 9, Varese 21100, Italy

A R T I C L E I N F O

Article history:

Received 13 May 2016

Received in revised form 11

September 2016

Accepted 16 September 2016

Available online

A B S T R A C T

Olive oil, a major feature of the Mediterranean diet, is an abundant source of phenolic com-

pounds. Olive oil production is associated with the generation of waste material, termed

‘olive mill wastewater’ (OMWW), that has been reported to be enriched in soluble polyphe-

nols. Given the known beneficial activity of polyphenols, we investigated whether the use

of purified extracts from OMWW, termed A009, rich in hydroxytyrosol, might have antican-

cer activities on colon cancer (CC) cell lines in vitro and in vivo and could represent a

chemopreventive preparation for CC. A009 from different batches inhibited proliferation,

migration, invasion, adhesion, sprouting of CC cells and release of angiogenic, pro-

inflammatory cytokines (VEGF, IL-8). Our data demonstrate that a novel purified, polyphenol

enriched extract, obtained from food industry waste material, with similar activity than pu-

rified hydroxytyrosol but easier to produce in large quantities and with an environment-

sensitive approach, has potential cancer chemopreventive properties for colon cancer cells.

© 2016 Published by Elsevier Ltd.

Keywords:

Olive mill wastewater

Polyphenols

Hydroxytyrosol

Chemoprevention

Colon cancer

* Corresponding author. Laboratory of Vascular Biology and Angiogenesis, Scientific and Technology Pole, IRCCS MultiMedica, Via G. Fantoli16/15, Milan 20138, Italy. Fax: +39 02 55406503.

E-mail address: [email protected] (A. Albini).1 These authors equally contributed.

Abbreviations: 7-AAD, 7-amino-actinomycin D; COD, chemical oxygen demand; CC, colon cancer; DAD, diode array detector; ECM, extra-cellular matrix; FACS, fluorescence-activated cell sorting; FSC, forward scatter; FITC, fluorescein isothiocyanate; 5-FU, 5-fluorouracil; FBS,foetal bovine serum; HPLC, high-performance liquid chromatography; HyT, hydroxytyrosol; LC, liquid chromatography; MS, mass spec-trometry; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide; OMWW, olive mill wastewater; PFA, paraformaldehyde;SSC, side scatter; UV–vis, ultraviolet–visible; VCR, volume concentration ratiohttp://dx.doi.org/10.1016/j.jff.2016.09.0091756-4646/© 2016 Published by Elsevier Ltd.

J o u rna l o f Func t i ona l F ood s 2 7 ( 2 0 1 6 ) 2 3 6 – 2 4 8

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1. Introduction

Epidemiological studies have shown a significant influenceof environment factors, lifestyle and diet in the insurgenceand in the prevention of pathologies associated with oxida-tive damage, such as cardiovascular diseases, diabetes,neurodegenerative conditions and cancer (Alarcon de la Lastra,Barranco, Motilva, & Herrerias, 2001; Lipworth, Martinez, Angell,Hsieh, & Trichopoulos, 1997; Sporn & Suh, 2000; Stark & Madar,2002; Stoneham, Goldacre, Seagroatt, & Gill, 2000; Trichopoulou,Lagiou, Kuper, & Trichopoulos, 2000; Tuck & Hayball, 2002). Ascancer represents a major cause of death (Jemal et al., 2008;Mozaffarian et al, 2015), there is currently an emphasis on ex-ploring approaches to prevent cancer incidence and mortality.

In the case of colon cancer (CC), lower rate of insurgencehas been observed amongst populations living within the Medi-terranean basin (Stark & Madar, 2002; Stoneham et al., 2000).Several studies have investigated dietary habits playing a rel-evant role in the prevention of CC. Olive oil represents a basiccomponent of the Mediterranean diet (Escrich, Moral, Grau,Costa, & Solanas, 2007; Rafehi et al., 2012; Widmer, Flammer,Lerman, & Lerman, 2015) and several data from both obser-vational and clinical studies showed that consumption of oliveoil is associated with reduced risk for chronic-degenerative dis-eases (InterAct Consortium, 2014; Prinelli et al., 2015;Schwingshackl & Hoffmann, 2014; Widmer et al., 2015).

Olive oil is a source of at least 30 phenolic compounds (Dais& Hatzakis, 2013). Amongst the phytochemicals present in oliveoil, phenols and their secoiridoid derivatives represent the mostabundant components that may be responsible for its preven-tive and protective effects. The concentrations and the relativeproportions of olive oil polyphenols depend on several factors,including the cultivars, the soil, the climate, the procedures foroil production and storage, and the degree of drupe matura-tion (Visioli, Bellomo, & Galli, 1998).The chemopreventive effectsof olive oil polyphenols have been investigated in in vitro models(Fabiani et al., 2002; Rosignoli, Fuccelli, Sepporta, & Fabiani,2016). Single phenols have been investigated for their molecu-lar properties; however, most of the studies concerning thebenefits associated with olive consumption have been focusedon the oil compartment of the fruit.

Large volumes of waste water (olive mill wastewater;OMWW) are generated during olive oil production, particu-larly during malaxation process (continuous washing of olivepaste with warm water prior to the procedure of separationof the oil from the paste) (Kanakis et al., 2013). OMWW is aproblematic and polluting effluent which alters soil and waterquality, with a relevant negative impact on ecosystemic func-tions, reducing chemical oxygen demand and leading toeutrophication (Justino et al., 2012). Altogether these issues rep-resent one of the major concerns for the olive oil industries,leading to an increase in disposal costs. An alternative use forOMWW is therefore highly auspicable.

Here, we investigated a polyphenol rich purified extract fromOMWW, termed A009, whose major phenolic components weredetermined. One of the most abundant polyphenols in A009was hydroxytyrosol (HyT). Other studies reported that OMWWcould be used to isolate polyphenols (Sedej et al., 2016;Vougogiannopoulou et al., 2015). The biological properties of

polyphenols include anti-oxidant, anti-apoptotic, anti-tumourand anti-inflammatory activities.

We tested the potential cancer chemopreventive activityof different batches of A009 as determined by inhibition of cellgrowth, adhesion, migration, sprouting and invasion andangiogenic, pro-inflammatory cytokine production, VEGFand IL-8, on two human colon-carcinoma cell lines (HT-29,HCT-116) and the murine colon-carcinoma cell line CT-26 invitro and confirmed in vivo using the CT-26 murine xenograftmodel.

2. Materials and methods

2.1. Reagents, chemicals and animals

The synthetic hydroxytyrosol (HyT), ≥98% in purity (Aglicon),was purchased from Cayman Chemicals (Ann Arbor, Michi-gan, USA). RPMI medium supplemented with 10% heat-inactivated foetal bovine serum (FBS) (Euroclone, Milan, Italy)and 1% glutamine (Euroclone, Milan, Italy), defined as com-plete medium, was used for cell line culturing and to inducemigration and invasion in related in vitro assays (Bruno et al.,2013). HPLC reagents, standards and solvents, were LC-MS gradeand purchased from Sigma-Aldrich. Standard stock solutionswere prepared by dissolving standard in a mixture ofmethanol:water, 90:10.

2.2. Cell cultures

Human colon cancer (CC) cell lines HT-29, HCT-116 and themurine CT-26 CC cell line where purchased from ATCC andgrown in RPMI medium (Sigma Aldrich Milan, Italy), supple-mented with 10% heat-inactivated foetal bovine serum (FBS)(Euroclone, Milan, Italy), 1% glutamine (Euroclone, Milan, Italy),1% penicillin–streptomycin at 37 °C in 5% CO2.

2.3. Preparation of A009 and phenolic quantification

OMWW were provided by Agriturismo La Vialla (CastiglionFibocchi, Arezzo, Italy) and used to obtain the phenol rich pu-rified extract A009 (Italian Patents n°1420804; n°1420805). Theexperiments were performed using four different batches ofA009 (A–D, Supplementary Table S1). A009 was obtained fromOMWW using two sequential cross-flow filtration processes aspreviously described (Rossi et al., 2015). Briefly, microfiltration(MF) was performed on a pilot plant equipped with tubularceramic modules membranes in alumina oxide with a MWCO0.45 micron. The MF permeate was further concentrated byreverse osmosis (RO) in a Polyamide spiral wound module(Microdyn Nadir, Wiesbaden, Germany) with a filtering surfaceof 7 m2. The RO permeate, representing ultrapure water, wasdiscarded. Finally, the RO concentrate, with a volume concen-tration ratio (VCR) of 3.6, constituted the olive extract (heretermed A009).

Phenolic composition of A009 was obtained by HPLC-DAD-MS-MS. Samples were analysed by HPLC (Supplementary Fig. S1)with UV–vis and MS (Supplementary Fig. S2) detection. Sampleextraction was performed as described previously in Zhang,

237J o u rna l o f Func t i ona l F ood s 2 7 ( 2 0 1 6 ) 2 3 6 – 2 4 8

Chen, Liang, & Liu (2006). Briefly, OMWW was homogenizedand 10 mL was added with 0.25 mL syringic acid as internalstandard (1.5 mg/mL). The solution was centrifuged (15 min,3200 × g), filtered (acetate cellulose filter, 0.45 µm, diameter30 mm) and diluted with pure Met-OH (methanol) to 25 mL.

The identification of phenolic compounds from samples wascarried out as previously reported (Klen, Wondra, Vrhovsek, &Vodopivec, 2015) by interpreting their mass spectra deter-mined via LC-MS-MS and comparing to data reported inliterature identified the compounds.The LC-MS-MS system con-sisted of a Shimadzu LCMS-8030 quadrupole mass spectrometer(Kyoto, Japan) operated in the electrospray ionization (ESI) mode,and a Shimadzu Nexera HPLC system (Kyoto Japan) whichconsisted of a degasser, two eluent pumps, a column ovenand an auto sampler. The separation was performed on areversed-phase Thermo Scientific Hypersil gold column (ODS250 × 4.6 mm, 3 micron), at room temperature.The mobile phaseconsisted of 1% aqueous formic acid (solvent A) and 1% formicacid in acetonitrile/methanol (25/75) (solvent B). Separation wasperformed using the following elution gradient: 2% B isocraticduring 10 min, from 2 to 98% B linear during 30 min, 98% Bisocratic during 7 min, then starting condition during 5 min tore-equilibrate the column. The flow rate was 0.6 mL/min, andthe injection volume was 10 µL. The column oven was set at30 °C. The auto-sampler was set at 15 °C and the auto-samplerneedle was rinsed before and after aspiration of the sampleusing methanol.

Mass spectrometry (MS) was performed acquiring spectraldata with the following ESI inlet conditions: nebulizing gas anddrying gas were nitrogen at a flow rate of 3.0 and 15.0 L/min,respectively; the interface voltage was set to −3.5 kV; desolvationline (DL) temperature was 250 °C and the heat block tempera-ture was 400 °C. The mass spectrometer operated in NegativeIon Scan and in Product Ion Scan mode using analyte-specificprecursor ions, with argon as the collision induced dissocia-tion gas (CID) at a pressure of 230 kPa. The characteristic UV–vis spectral shapes and MS fragmentation profiles of knownand phenol components were attributed according to a pre-vious work (Klen et al., 2015). HPLC analysis was performedusing a diode-array detector liquid chromatographic system(model 1050), equipped with an auto-sampler (model 1100,Agilent Technologies, Palo Alto, CA, USA). The software usedwas Agilent Chemstation. A Thermo Scientific Hypersil goldcolumn (ODS 250 × 4.6 mm, 3 µm), equipped with a Thermo Sci-entific pre-column (C18 ODS 4.0/4.6 mm), was used.

The injection volume was 20 µL. Elution was performed ata flow rate of 0.8 mL min−1 using a methanol (solvent A), acidwater (0.2% orthophosphoric acid) (solvent B), acetonitrile(solvent C) mixture as a mobile phase. Quantification re-vealed that the major phenolic component of A009 is HyT, alongwith several other phenolic compounds (SupplementaryTable S1).

All these results were complemented with the UV spectraprovided by DAD (Diode Array Detector) in terms of the ab-sorbance bands.

2.4. In vitro cell proliferation

HT-29, HCT-116, CT-26 (103 cells/well) were plated into 96-well plate in RPMI supplemented with 10% heat-inactivated

foetal bovine serum (FBS) (Euroclone), 1% glutamine (Euroclone),1% penstrept (Euroclone) at 37 °C in 5% CO2 and left to adhereovernight. Cell proliferation was assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide;Sigma Aldrich, Milan, Italy) assay. Following cell adhesion,treatments were performed with decreasing dilutions (rangingfrom 1:50 to 1:10000) of A009, or HyT, prepared in complete RPMImedium. The HyT solution (70% EtOH:30% water) was pre-pared for each experiment to obtain the same concentrationof HyT present in the relative A009 batch, finally diluted in RPMIcomplete medium. Multiple final dilutions were used.

Reference HyT was 1.75 × 10−2 M for batch A, 3.71 × 10−2 M forbatch B, 3.57 × 10−2 M for batch C and 1.63 × 10−2 M for batch D(Supplementary Table S1). 70% EtOH, further diluted in RPMIcomplete medium, was used as a negative control (solvent).Cells were treated for 24–96 h and the relative absorbance wasmeasured at 570 nm by a FLUOstar spectrophotometer(FLUOstar Omega BMG LABTECH, Ortenberg, Germany).

2.5. Detection of apoptosis in vitro

HT-29, HCT-116, CT-26 (2 × 105 cells/well) were plated into 6-wellplates and exposed to decreasing dilutions (1:500, 1:250) of A009,HyT or EtOH for 24 h and 48 h in RPMI supplemented with 10%FBS (Euroclone, Milan, Italy), 1% glutamine (Euroclone) and 1%penicillin–streptomycin (Euroclone) at 37 °C in 5% CO2. Cellswere recovered, washed twice with PBS, transferred to 5 mLpolystyrene tubes (BD Biosciences Milan, Italy). Cells were re-suspended in AnnexinV-binding buffer (BD Biosciences, Milan,Italy) and stained with fluorescein isothiocyanate (FITC)-conjugated AnnexinV and 7-amino-actinomycin D (7-AAD) (BDBiosciences Milan, Italy) for 15 min at 4 °C in the dark. Cellswere then washed in PBS, supernatants were discarded andresuspended in 400 µL of PBS. Analysis was performed usinga FACSCantoII flow cytometer (BD Biosciences). Physical pa-rameters FSC/SSC were used to exclude cell debris and the rateof dead cells was determined as Annexin+7AAD−/+ events. Datawere analysed using FACSDiva Software 6.1.2.

2.6. Cell adhesion assay in vitro

Adhesion assay was performed as previously described (Carregaet al., 2015). Cells were incubated for 24 h as indicated above.After treatment, 3 × 103 cells were seeded on 4-well chamberslides pre-coated for 45 min with 2 µg/mL fibronectin (SigmaAldrich). Following 90 min incubation, the supernatants wereremoved and cells were washed with PBS. Cells were fixed with4% paraformaldehyde (PFA) and stained with DAPI (SigmaAldrich). Assays were performed in triplicates wells. Five mi-croscope fields were randomly selected from the wells for eachtreatment to count the number of adherent cells and the datawere analysed using analysis of variance (ANOVA).

2.7. Migration and invasion assays in vitro

Migration and invasion assays were performed using a modi-fied Boyden chambers, as previously described (Albini & Benelli,2007; Albini et al., 1987; Albini, 2016). HT-29, HCT-116, CT-26 cells(5 × 104), treated with A009 or CTRL for 24 h, were washed withPBS, resuspended in serum-free medium and placed in the

238 J o u rna l o f Func t i ona l F ood s 2 7 ( 2 0 1 6 ) 2 3 6 – 2 4 8

upper compartment of the Boyden chamber. RPMI medium,supplemented as described in the Materials and Methods sectionwas used as a chemoattractant and added in the lower com-partment. 10 µm pore-size polycarbonate filters, pre-coated withMatrigel (1 mg/mL, BD) for chemoinvasion assay and with col-lagen IV (50 µg/mL, Sigma Aldrich) for chemotaxis assay wereused as the interface between the two chamber compart-ments. Following 6 h (chemotaxis) or 24 h (chemoinvasion) ofincubation at 37 °C in 5% CO2, the filters were recovered, cellson the upper surface were mechanically removed and mi-grated or invaded cells on the lower filter surface were fixedwith absolute ethanol and stained with DAPI. Cells were countedin a double-blind manner in 5 consecutive fields with a ZeissMicroscope associated with a Nikon camera.

2.8. In vitro sprouting assay

The sprouting assay was performed using 48 well plates coatedwith Matrigel. HCT-116 and CT-26 (4 × 104) cells were seededinto the Matrigel and immediately treated with A009 or CTRLfor 24 h, as described above. The formation of sprouts wasobserved at 24 h using an inverted microscope at 10× magni-fication. Assays were performed in triplicate wells. Fifteenmicroscope fields were randomly selected from three wells foreach treatment to count the number of sprouts/cell and thedata were analysed using analysis of variance (ANOVA).

2.9. Analysis of cytokine production

The effect of cytokine production by A009 treated CC cell lineswere determined by Flow Cytometry. HCT-116 cells were treatedwith increasing dilution (1:250, 1:500) of A009 or CTRL for24 h. Golgi stop agent Monensin (2 mg/L, Sigma) was addedduring the treatment. Cells were detached, washed in PBS,permeabilized using the Cytofix/Cytoperm reagent and finallystained for intracellular cytokines using PE-conjugated mono-clonal antibodies against VEGF and IL-8 (R&D System,Minneapolis, MN). Cytokines were measured in a FACSCantoIIflow cytometer (BD), as already described (Solinas et al., 2012).

2.10. Tumour cell growth in vivo

The effect of A009 or HyT on tumour cell growth was as-sessed using the CT-26 colon carcinoma cell line in syngenicBalbC mice. The use of animals was in accordance with theItalian and European Community guidelines (D.L. 2711/92No.116; 86/ 609/EEC Directive). The procedure was approved bythe local animal experimentation ethics committee (Univ.Insubria ID#05/13) and by the Italian Health Ministry. 106 CT-26 cells per animal, suspended in 100 µL of liquid pre-chilledMatrigel (10 mg/mL, BD) were subcutaneously injected into theright flank of each mouse. Treated animals (8 per group) re-ceived intraperitoneally (i.p.) injection of A009 or HyT at 1:500or 1:250 dilutions. PBS and 5-fluorouracil (5-FU; 10 mg/kg every2 days, 1 mg/kg every day) were used as negative and posi-tive controls, respectively. We also tested the effects of A009administered orally (per os) using A009 or HyT at 1:100 or 1:50in drinking water.Two days prior and two days following tumourcell injection, treatments were administered via i.p. or per os.

Tumour xenograft growth rates were monitored every 2 dayswith a caliper and determined using the rational ellipse formula:

Tumour volume width length= × ×( )2 0 5. . (1)

The health state of the animals (food and water consump-tion, dejections, skin conditions, reactivity to stimuli) wasmonitored during the experimental procedures. At the end ofthe experiment, tumours were excised and weighed.

2.11. Statistical analyses

The statistical significance between multiple data sets was de-termined by one-way ANOVA using Graph-Pad PRISM. Tumourgrowth curves were determined by two-way ANOVA. FACS datawere analysed by FACSDiva Software 6.1.2. Data are ex-pressed as means ± SEM.

3. Results

3.1. A009 inhibits the proliferation of colon cancer cells

A009 (dilution range 1:50 to 1:10,000) from 4 different batchesmade from different OMWW were tested for its ability to in-terfere with tumour cell growth by MTT assay on two human(HT-29, HCT-116) and one murine (CT-26) colon-cancer cell lines.Total phenol composition of the four diverse batches of A009was determined by HPLC analysis, showing similar composi-tion in phenols (Supplementary Table S1) with some differences.As shown in Supplementary Table S1, the most abundant poly-phenol is the HyT, which, for this reason, was chosen asreference compound, at the same concentration contained inthe relative batch.

The effect of A009 on cell proliferation was compared withthe HyT alone. A009 (Fig. 1, Batch A) was able to inhibit HT-29cell proliferation from 48 h following treatment, showing aslower growth rate, starting from the 1:500 dilution (Fig. 1A).A009 inhibited growth also of human HCT-116 (Fig. 1B) andmurine CT-26 (Fig. 1C) cell lines. Batches B, C, D behaved in thesame way as shown in Supplementary Fig. S1.The four batchesshowed comparable effects in reducing cell proliferation(batches B, C and D, shown in Supplementary Fig. S3 a, b, c re-spectively for HT-29, HCT-116 and CT-26 cells). We found thatthe effects of A009 on cell proliferation were similar to thoseexhibited by HyT alone at the same dilution relative to the batchused. The lowest (1:50) dilution of A009 that arrested cell pro-liferation in the three cell lines was further investigated. Thiswas not due to toxicity. No significant pro-apoptotic effect wasobserved in HT-29, HCT-116, CT-26 cell lines, treated with A009batch A (Fig. 2) as well as with batches B, C, D (SupplementaryFig. S4A, B, C, respectively for HT-29, HCT-116 and CT-26 cells).Given the comparable activities, one of the A009 batches (batchC) and the lowest active dilutions (1:500 and 1:250) were se-lected for the remaining experiments.

3.2. A009 impairs the adhesion of colon cancer cells invitro

Tumour cell adhesion to ECM represents a key step in the meta-static cascade (Albini, 2016; Seyfried & Huysentruyt, 2013). Using


HCT-116

A

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NT1:100001:50001:10001:5001:2501:1001:50

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RPMI + SOLVENT RPMI + HyT (2,7 g/L) RPMI + A009 (BATCH A)



NT: not treatedHyT: hydroxytyrosolSolvent: ethanol 70%/water 30%A009: purified polyphenol enriched extract from olive mill waste water

Fig. 1 – Anti-proliferative properties of A009 on human and murine CC cells. Cell proliferation was evaluated at sequentialtime points (24, 48, 72, 96 h) using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. A009(batch A) and HyT (hydroxytyrosol) inhibit proliferation of both human (A, B) and murine (C) CC cells in a concentration-dependent manner. Results are shown as mean ± SEM. The experiment was performed also with a negative control.Solvent (70% ethanol) is shown as a negative control. Batches B, C and D behaved similarly (Supplementary figure S1).


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NT: not treated; HyT: hydroxytyrosol; Solvent: ethanol 70%/water 30%, A009: purified polyphenol enriched extract from olive mill waste water

Fig. 2 – Induction of apoptosis in human and murine CC cell lines treated with A009. Effect of A009 (batch A) and HyT(hydroxytyrosol) on human (A and B) and murine (C) CC cell survival. Cell apoptosis was assessed by flow cytometry andviable cells are described as Annexin V-/7-AAD- cells. No apoptosis induction was observed after 24 h and 48 h oftreatment with A009. Untreated (NT) and solvent (70% ethanol) were used as negative controls. Mean ± SEM of 3independent experiments is shown.


fibronectin as an ECM layer, we evaluated the ability of A009(batch C) to interfere with the attachment of HT-29, HCT-116and CT-26 cells that were pre-treated for 24 h with A009 (1:500,1:250) or solvent (EtOH 70%/30% water diluted in RPMI). A009and HyT significantly impaired HCT-116 (Fig. 3A), HT-29 (Fig. 3B)and CT-26 (Fig. 3C) adhesion.

3.3. A009 inhibits migration and invasion of coloncancer cells in vitro

The migratory and invasive abilities of cancer cells are criti-cal processes of the metastatic cascade (Albini, 2016). Mostmalignant tumour cells show a chronically invasive pheno-type on the basis of activation of specific cellular and molecularsignalling pathways. We investigated the capability of A009 toprevent cell migration (Fig. 4) and invasion (Fig. 5) at 24 h. Asshown in Figs. 4 and 5, A009 (batch C) and HyT significantlyinhibited the migration and invasion capabilities of HCT-116(Figs. 4 and 5A), HT-29 (Figs. 4 and 5B) and CT-26 (Figs. 4 and5C) CC cells.

3.4. A009 reduces sprout formation of colon cancer cellsin vitro

Sprouting capabilities on matrix have been described for cancercells and are associated with their invasive properties (Terzuoliet al., 2010). Here, we evaluated the ability of A009 (batch C)to prevent sprouting formation of HCT-116 and CT-26 CC cellson a basement membrane matrix (Matrigel). A009 and HyT sig-nificantly reduced sprout formation in HCT-116 (Fig. 6A) andCT-26 (Fig. 6B) cells.

3.5. A009 inhibits VEGF and IL-8 levels in vitro

Previous findings reported that HyT had strong anti-inflammatory/anti-angiogenic activities and blocks theexpression of VEGF (Lamy, Ouanouki, Beliveau, & Desrosiers,2014; Terzuoli et al., 2010). Production of VEGF is associated withthe capability of cancer cells to stimulate tumour angiogen-esis (Albini et al., 2015; Arjaans et al., 2016; Albini, Tosetti, Li,Noonan & Li, 2012). Here, we examined the effects of A009 oninhibition of VEGF and IL-8 expression by HCT-116 cells. Wefound that A009 (batch C) and HyT significantly reduced theproduction of VEGF (Fig. 7A) and IL-8 (Fig. 7B) protein. This isconsistent with an anti-angiogenic activity of A009 and HyT.

3.6. A009 interferes with tumour cell growth in vivo

We investigated in vivo the ability of A009 to interfere with sub-cutaneously injected murine CT-26 colon cancer cell growthusing the syngenic BalbC murine model. In one experiment,the mice were treated i.p. either with A009 (batch C) or HyT(1:500, 1:250). In another experiment, the mice were treated withA009 (batch A) or HyT (1:100, 1:50) per os in the drinking water.The treatment started two days prior tumour cell injection andthe mice were treated every two days during the experiment.As positive control, we used i.p. 10 mg/mL 5-FU, a commonlyemployed chemotherapeutic agent for colon cancer (Albini et al.,2010; Goldstein, Zeichner, Bartnik, Neustadter, & Flowers, 2016;Sinicrope, Okamoto, Kasi, & Kawakami, 2016), administered

every 2 days following tumour challenge. PBS was used as anegative control. In the i.p. setting, the lowest dilution of HyTdid not show a significant effect, while both concentrations ofA009 showed significant (p < 0.001 and p < 0.0001, respec-tively for A009 1:500 and A009 1:250) reduction of tumour growth(Fig. 8A). In the oral treatment setting (per os), we found thatA009 (1:100, 1:50) inhibited CT-26 tumour growth (p < 0.0001 forboth A009 1:500 and A009 1:250) while the same effect was ob-served only for the less diluted HyT (1:50) preparation (Fig. 8B).

Tumour weight at sacrifice was also assessed. Tumoursexcised from mice treated with A009 exhibited a smaller size(Supplementary Fig. S5A), compared to PBS-treated mice andsimilar to those excised from HyT-treated mice. Assessmentof tumour weight revealed that A009 (1:500) and HyT (1:250 atthe same concentration of the related A009 batch), limitedtumour weight in the per os schedule (Supplementary Fig. S5Band C).

4. Discussion

Chemoprevention, as first defined by Michael Sporn in 1976,uses natural or synthetic agents to reverse, suppress, or preventcarcinogenesis, delaying progression to invasive cancer (Sporn& Suh, 2000). Chemoprevention by dietary phytochemicals isparticularly attractive for their potential low toxicity and fortheir ability to modulate a plethora of signal transduction path-ways in several biological processes associated with cancer,inflammation and angiogenesis (Albini, Tosetti, Li, Noonan, &Li, 2012; Landis-Piwowar & Iyer, 2014; Ferrari et al., 2011; Sognoet al., 2009; Sporn & Suh, 2000).

Olive oil, a major feature of the Mediterranean diet, is anabundant source of chemopreventive phenolic compounds.Olive oil production is associated with the generation of wastematerial, termed ‘olive mill wastewaters’ (OMWW), that hasbeen reported to be enriched in soluble polyphenols (Justinoet al., 2012), thus representing an extremely attractive sourceof nutraceutical products. Currently, there is a growing inter-est in studying phenolic compounds in OMWW representingan aqueous phase enriched in soluble phenols. However, fewstudies have focused on the biological effects of this wasteproduct, especially in the context of cancer prevention.One of the most abundant polyphenol in OMWW is HyT(Vougogiannopoulou et al., 2015). The biological properties ofpolyphenols include anti-oxidant, anti-apoptotic, anti-tumourand anti-inflammatory activities. HyT is able to inhibit bothinitiation and promotion/progression phases of carcinogen-esis by preventing DNA damage induced by different genotoxicmolecules and by inhibiting proliferation and inducing apop-tosis in different tumour cell lines (Acquaviva et al., 2012; Fabianiet al., 2008; Goulas et al., 2009; Owen et al., 2004; Rosignoli et al.,2016). Chemopreventive effects of HyT have also been ob-served in HL60 human promyelocytic leukaemia cells, HT-29and DLD1 colon adenocarcinoma cells, reducing cell prolifera-tion due to induction of apoptosis (Achmon & Fishman, 2015;Corona et al., 2009; Sun, Luo, & Liu, 2014; Terzuoli et al., 2010).Oleuropein is also a component of A009. Recent data demon-strated that oleuropein can induce apoptosis through thedownregulation of pAkt, suggesting an inhibitory effect on Akt


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Fig. 3 – Effects of A009 on human and murine CC cell lineadhesion. A009 (batch C) and HyT (hydroxytyrosol) wereable to inhibit CC adhesion compared to untreated cells NT.Representative pictures show adherent HT-29 (A), HCT-116(B) and CT-26 (C) cells (magnification 10×). Results areshown as mean ± SEM of 5 different fields captured inthree experiments. ANOVA statistical analysis was used todetermine p-value (***p < 0.001). Untreated (NT) and solvent(70% ethanol) were used as negative controls.

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Fig. 4 – Effects of A009 on human and murine CC cell linemigration. The ability of A009 (batch C) and HyT(hydroxytyrosol) to interfere with human and murine cellline migration was assessed using a modified Boydenchamber assay compared to untreated cells (NT).Representative pictures show migrated HT-29 (A), HCT-116(B) and CT-26 (C) cells (magnification 10×). Results areshown as mean ± SEM of 5 different fields captured inthree experiments. ANOVA statistical analysis was used todetermine p-value (**p < 0.01, ***p < 0.001). Untreated (NT)and solvent (70% ethanol) were used as negative controls.


signalling (Acquaviva et al., 2012). Oleuropein can increasetrastuzumab efficacy in breast cancer cells (Fayyaz et al., 2016).Amongst the other soluble polyphenols, verbascoside hasbeen reported to exert anti-proliferative activities towardstumour cells in vitro (Wartenberg et al., 2003), to induceapoptosis by telomere–telomerase cell cycle-dependentmodulation, and to enhance repair of DNA damage due to byoxidative stress. Verbascoside and natural plant-derived poly-phenols (phenylethanoids, resveratrol) have been reported to

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Fig. 5 – Effects of A009 on human and murine CC cell lineinvasion. The ability of A009 (batch C) and HyT(hydroxytyrosol) to interfere with human and murine cellline invasion was assessed using a modified Boydenchamber assay compared to untreated cells NT.Representative pictures show invaded HT-29 (A), HCT-116(B) and CT-26 (C) cells (magnification 10×). Results areshown as mean ± SEM of 5 different fields captured inthree experiments. ANOVA statistical analysis was used todetermine p-value (**p < 0.01, ***p < 0.001). Untreated (NT)and solvent (70% ethanol) were used as negative controls.

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Fig. 6 – Effects of A009 on human and murine CC cellsprouting. The ability of A009 (batch C) and HyT(hydroxytyrosol) to interfere with human HCT-116 (A) andmurine CT-26 (B) cell sprouting on Matrigel was evaluatedcompared to untreated cells (NT). Results are shown asmean ± SEM of 5 different fields captured in threeexperiments. ANOVA statistical analysis was used todetermine p-value (**p < 0.01, ***p < 0.001). Untreated(NT) and solvent (70% ethanol) were used as negativecontrols.


synergize with conventional anti-cancer therapies (Alipieva,Korkina, Orhan, & Georgiev, 2014).Anti-tumour effects of olivederivatives have been reported for cancers affecting differentorgans, including pancreas, oral cavity, oesophagus, prostateand lung (Rafehi, Ververis, & Karagiannis, 2012).

Colon cancer (CC) is the second most common cancer inwomen and third most common cancer in men worldwide(El Zoghbi & Cummings, 2016). Lifestyle and dietary habitshave been reported to significantly impact on coloncancer prevention (Vipperla & O’Keefe, 2016; Vulcan et al.,

2015), suggesting the importance of nutrition in cancer(chemo)prevention.

Here we investigated the effects of a novel purifiedpolyphenol-enriched extract of OMWW, A009, abundant in HyTbut also containing other polyphenols (Supplementary Table S1),on human and murine colon cancer cells. Chemical charac-terization of the extracts confirmed the presence of polyphenols,as demonstrated by the HPLC analysis, and the abundance ofHyT, shown by HPLC and MS data. In the initial assessmentof the ability of A009 to inhibit cancer cell growth in vitro, wetested four different batches of the extract and compared themto HyT at similar concentrations in a dose and time-dependantmanner. We found that all batches of A009 inhibited coloncancer cell proliferation in a similar manner, and these effectswere not associated to induction of apoptosis.

Since tumour cell motility, invasion, and metastasis todistant sites (Albini, 2016) represent crucial hallmarks of cancer(Hanahan & Weinberg, 2011), we evaluated the effects of A009on colon cancer cell ability to adhere, migrate and invade. Giventhat all the batches behaved similarly, we chose batch C forinvestigation of these parameters. We found that incubationof cells with A009 for 24 h significantly inhibited HCT-116, HT-29 and CT-26 adhesion, invasion and migration.These data werefurther supported by the ability of A009 to inhibit CT-26 sprout-ing on a basement membrane matrix (Matrigel). Given thepreviously reported anti-inflammatory effects of HyT (Lamyet al., 2014; Terzuoli et al., 2010), we examined the ability ofA009 to prevent the production of pro-inflammatory and an-giogenic cytokines, VEGF and IL-8. Only viable cells, accordingto FSC/SSC parameters, have been considered. A009 (batch C)significantly (p < 0.01 and p < 0.001, respectively for A009 1:500and 1:250) reduced VEGF and IL-8 protein expression in HCT-116 cells. All these effects were similar to those exerted bycomparable concentrations of HyT. A009, which is easier toproduce and less costly than HyT and is a waste product ofthe food industry, could be therefore a valid and environmen-tally friendly way to administer polyphenol, HyT-richpreparations.

Finally, the effect of A009 on tumour cell growth was as-sessed using the CT-26 colon-carcinoma cell lines in 6–8 weeksold syngenic BalbC mice, and compared to the effects of HyTand a well-known colon cancer chemotherapeutic drug, the5-FU. A009 was able to inhibit tumour cell growth in a dose-dependent manner, exerting an inhibitory effect starting fromthe lowest dilution whereas purified HyT reduced tumourgrowth only at the highest dose. These data were further cor-roborated by the evaluation of weight of tumours excised fromtreated mice. These results suggest that the A009 extract hasadditional active components relative to commercially avail-able HyT.

5. Conclusions

Taken together, our results suggest that isolating a purified frac-tion of the waste material OMWW (A009) represents a promisingstrategy to both limit environment pollution and obtain a po-tential water soluble polyphenol rich nutraceutical product with

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beneficial effects associated with potential chemopreventiveproperties for colon cancer.

Authors’ contributions

Conceived and designed the experiments: AA, DMN, AB. Per-formed the in vitro experiments: BB, DDS, AB,TR, NM. Performedthe in vivo experiments: AB, BB. Analysed the data: AA, DMN,AB, DDS, BB, NM, PC. Wrote the paper: AA, DMN, AB, DDS, BB.Purified the A009 extracts and provided their characteriza-tion: DP.

Acknowledgements

This study was supported by the Fattoria La Vialla di Gianni,Antonio e Bandino Lo Franco – SAS and by theAIRC,AssociazioneItaliana Ricerca sul Cancro IG14600 to AA. Massimo Pizzichiniand DP (Genelab srl,ENEA) generated theA009 extracts.We thankMarzia Migliorini, Divisione Laboratorio Chimico Merceologico,for the chromatography analyses and Simone Cristoni (I.S.B. –Ion Source & Biotechnologies) for the mass spectroscopy analy-sis. BB andTR are students of the PhD program in Biotechnology,Biosciences and Surgical Technologies, University of Insubria.AB was a FIRC (Fondazione Italiana per la Ricerca sul Cancro)fellow and is currently a fellow for Fondazione UmbertoVeronesi(FUV). We like to thank Alessandra Panvini Rosati for secretaryassistance. The funding agencies had no influence on the ex-perimental methods and interpretation of the data.

Appendix: Supplementary material

Supplementary data to this article can be found online atdoi:10.1016/j.jff.2016.09.009.

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Fig. 8 – Anti-tumour effects of A009 in vivo. CT-26 (106) per animal, suspended in 100 µL of liquid pre-chilled Matrigel(10 mg/mL, BD) was subcutaneously injected into the right flank of each mice. Tumour dimension was measured with acaliper for 12 days after injection. (A) Tumour growth in mice treated i.p. with A009 (Batch C), HyT (hydroxytyrosol) orthe reference drug 5-fluorouracil (5-FU). (B) Tumour growth in mice treated per os (drinking water-Batch A) with A009or HyT. The mean ± SD is shown (***p < 0.001; ****p < 0.0001).


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Potential chemopreventive activities of a polyphenol rich purified extract from olive mill wastewater on colon cancer cells Introduction Materials and methods Reagents, chemicals and animals Cell cultures Preparation of A009 and phenolic quantification In vitro cell proliferation Detection of apoptosis in vitro Cell adhesion assay in vitro Migration and invasion assays in vitro In vitro sprouting assay Analysis of cytokine production Tumour cell growth in vivo Statistical analyses

Results A009 inhibits the proliferation of colon cancer cells A009 impairs the adhesion of colon cancer cells in vitro A009 inhibits migration and invasion of colon cancer cells in vitro A009 reduces sprout formation of colon cancer cells in vitro A009 inhibits VEGF and IL-8 levels in vitro A009 interferes with tumour cell growth in vivo

Discussion Conclusions Authors' contributions Acknowledgements Supplementary material References

potential chemopreventive activities of a polyphenol rich ......potential chemopreventive activities...

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