-
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
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/ locate / j ff
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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
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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
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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
239J o u rna l o f Func t i ona l F ood s 2 7 ( 2 0 1 6 ) 2 3 6
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HCT-116
A
B
NT1:100001:50001:10001:5001:2501:1001:50
Time (h)
OD
570
nm
0 24 48 72 960.00.30.60.91.21.51.82.12.42.73.0
0 24 48 72 960.00.30.60.91.21.51.82.12.42.73.0
0 24 48 72 960.00.30.60.91.21.51.82.12.42.73.0
CT-26
0 24 48 72 960.00.30.60.91.21.51.82.12.42.73.0
0 24 48 72 960.00.30.60.91.21.51.82.12.42.73.0
0 24 48 72 960.00.30.60.91.21.51.82.12.42.73.0
C
HT-29
0 24 48 72 960.00.30.60.91.21.51.82.12.42.73.0
0 24 48 72 960.00.30.60.91.21.51.82.12.42.73.0
0 24 48 72 960.00.30.60.91.21.51.82.12.42.73.0
RPMI + SOLVENT RPMI + HyT (2,7 g/L) RPMI + A009 (BATCH A)
RPMI + SOLVENT RPMI + HyT (2,7 g/L) RPMI + A009 (BATCH A)
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).
240 J o u rna l o f Func t i ona l F ood s 2 7 ( 2 0 1 6 ) 2 3 6
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:250
A009
1:500
A009
1:250
APOPTOSIS CT-26
APOPTOSIS HCT-116
APOPTOSIS HT-29
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.
241J 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
-
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
242 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
-
A
B
C
HCT
-116
HT-
29CT
-26
NT Solvent Hyt A009 Solvent Hyt A009 1:500 1:500 1:500 1:250
1:250 1:250
0
10
20
30
40
50
****** ******
Num
ber
of a
dher
ent c
ells
0
40
80
120
*** ******
***
Num
ber
of a
dher
ent c
ells
0
20
40
60
80
****** ******
Num
ber
of a
dher
ent c
ells
NT: not treatedHyT: hydroxytyrosolSolvent: ethanol 70%/water
30%, A009: purified polyphenol enriched extract from olive mill
waste water
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.
A
B
C
NT Solvent Hyt A009 Solvent Hyt A009 1:500 1:500 1:500 1:250
1:250 1:250
0
50
100
150
200
250
****** *** ***Num
ber
of m
igra
ted
cells
HCT
-116
HT-
29CT
-26
0
40
80
120
***
***
***
***
Num
ber
of m
igra
ted
cells
0
50
100
150
200
250
** **
*****
Num
ber
of m
igra
ted
cells
NT: not treatedHyT: hydroxytyrosolSolvent: ethanol 70%/water
30%, A009: purified polyphenol enriched extract from olive mill
waste water
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.
243J 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
-
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
0
20
40
60
80
100
*** *** *** ***
Num
ber
of in
vade
d ce
lls
0
20
40
60
80
100
****** *** ***
Num
ber
of in
vade
d ce
lls
0
20
40
60
80
*** ********
Num
ber
of in
vade
d ce
lls
NT Solvent Hyt A009 Solvent Hyt A009 1:500 1:500 1:500 1:250
1:250 1:250
A
B
C
HCT
-116
HT-
29CT
-26
NT: not treatedHyT: hydroxytyrosolSolvent: ethanol 70%/water
30%, A009: purified polyphenol enrichedextract from olive mill
waste water
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.
HCT 116
CT26B
A
0
5
10
15
** ** ******Nu
mbe
r of
spr
outs
0
5
10
15
20
25
*****
*****
Num
ber
of s
prou
ts
HCT-116
CT-26
NT: not treatedHyT: hydroxytyrosolSolvent: ethanol 70%/water
30%, A009: purified polyphenol enriched extract from olive mill
waste water
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.
244 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
-
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
HCT-116A
0
2
4
6
** ** ****% o
f IL-
8+ c
ells
0
20
40
60
80
** ** ******
% o
f VEG
F+ c
ells
NT: not treatedHyT: hydroxytyrosolSolvent: ethanol 70%/water
30%, A009: purified polyphenol enriched extract from olive mill
waste water
B HCT-116
Fig. 7 – Effects of A009 on VEGF and IL-8 expression in HCT-116
cells. FACS analysis shows the expression of VEGF (A)and IL-8 (B)
in HCT-116 cells compared to untreated cellsNT in HCT-116 cells
treated with A009 (batch C) or HyT(hydroxytyrosol). Results are
shown as mean ± SEM.Statistical analysis was used to determine
p-value(**p < 0.01; ***p < 0.001). Untreated (NT) and solvent
(70%ethanol) were used as negative controls.
245J 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
-
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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TUMOR GROWTH PER OS
0 2 4 6 8 10 120
250
500
750
1000
1250
1500PBSA009 1:50Hyt 1:50A009 1:100Hyt 1:100
********
****
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DAYS
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5-fluorouracilA009: purified polyphenol enriched extract from olive
mill waste waterIP: intraperitoneal
BA
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0.001; ****p < 0.0001).
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248 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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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