oods
A icle
Ohmic Hea ing Ex ac o Vine P uning Residue Has
An i-Colo ec al Cance Ac i i y and Inc eases
Sensi i i y o he Chemo he apeu ic D ug 5-FU
Mei ielly S. Jesus 1,2,†, Ana C. Ca alho 2,3,†, JoséA. Teixei a 1, Lucília Domingues 1and
C is ina Pe ei a-Wilson 1,2,*
1CEB–Cen e o Biological Enginee ing, Uni e si y o Minho, 4710-057 B aga, Po ugal;
[email p o ec ed] (M.S.J.); [email p o ec ed] (J.A.T.); [email p o ec ed] (L.D.)
2Depa men o Biology, Uni e si y o Minho, 4710-057 B aga, Po ugal; [email p o ec ed]
3CITAB–Cen e o he Resea ch and Technology o Ag o-En i onmen al and Biological Sciences,
Uni e si y o T ás-os-Mon es and Al o Dou o, 5000-801 Vila Real, Po ugal
*Co espondence: [email p o ec ed]; Tel.: +351-253604318; Fax: +351-253604319
†These au ho s con ibu ed equally o his wo k.
Recei ed: 30 June 2020; Accep ed: 10 Augus 2020; Published: 12 Augus 2020


Abs ac :
Vine p uning esidues a e by-p oduc s o he wine indus y ha ha e no ecei ed much
a en ion in he pas , in spi e o being ich in bioac i e compounds. In his s udy, we aimed o es
whe he an ohmic ex ac o ine p uning esidue (VPE) has an i-colo ec al cance (CRC) p ope ies,
and whe he esponses di e acco ding wi h cell’s mu a ion p o ile. VPE dec eased human CRC
cell p oli e a ion, accompanied by DNA e ec s and cell cycle modula ion. VPE also inc eased
cell sensi i i y o he chemo he apeu ic d ug 5-FU. Ou esul s sugges ha umo s ha bo ing
BRAF mu a ions may be mo e esponsi e o VPE han KRAS mu a ed umo s. These e ec s o
he ex ac we e no comple ely ep oduced by he mos abundan cons i uen s es ed indi idually
a he concen a ions p esen in he e ec i e dose o VPE. Globally, ou esul s indica e ha VPE,
a polyphenol en iched ex ac p oduced by ohmic hea ing o ine p uning esidue, has an i-colo ec al
cance po en ial, including sensi izing o a chemo he apeu ical d ug, and i s use in unc ional oods o
nu aceu icals could be exploi ed in pe sonalized an i colo ec al cance die a y s a egies. Valo iza ion
o his lignocellulosic esidue should encou age bio-was e ecycling, adding alue o his ag icul u al
by-p oduc and p omo ing he sus ainable use o na u al esou ces.
Keywo ds:
ine p uning esidue; bio-was e alo isa ion; ohmic polyphenol ex ac ion; colo ec al
cance ; 5-FU; RKO cells; HCT116 cells; apigenin; que ce in; unc ional oods
1. In oduc ion
En i onmen al conce ns ha e p omp ed he desi e o educe he impac o he ag o- ood indus y,
and ans o m oday’s linea economy in o a mo e e icien ci cula model. In line wi h his, he scien i ic
communi y has been de o ing majo e o s o he sea ch o bio-was e ecycling me hodologies ha
p omo e sus ainable g ow h, based on he e icien use o na u al esou ces [1,2].
G apes a e a majo ag icul u al p oduc , and acco ding o he 2019 S a is ical Repo on Wo ld
Vi i inicul u e [
3
], 77.8 million ons we e p oduced wo ldwide in 2018, wi h 57% being used o wine
making. The Eu opean Union (EU) alone accoun s o 45% o he wo ld’s a ea unde ine, co esponding
o 3.2 million hec a es [
4
]. Po ugal is wi hin he EU’s op i e wine p oduce s (conside ing li es
p oduced pe land squa e kilome e), and has he ou h la ges ag icul u al a ea unde ine a e
Spain, F ance, and I aly [3].
Foods 2020,9, 1102; doi:10.3390/ oods9081102 www.mdpi.com/jou nal/ oods
Foods 2020,9, 1102 2 o 11
Many g ape ine by-p oduc s, such as g ape pomace, seeds, s ems, and ine lea es, ha e been
ex ensi ely cha ac e ized in e ms o chemical composi ion and biological p ope ies [
5
,
6
], and ha e
been used as ing edien s in die a y supplemen s [
7
] and cosme ics [
8
]. Vine p uning esidues, howe e ,
emain unde -exploi ed, and a e o en bu ned in he ields, con ibu ing o g een-house gas emissions,
which uns coun e o he u gen need o clima e change mi iga ion [9,10].
Vine p uning esidues cons i u e aluable biomass ha can be used in a bio e ine y scheme,
conside ing hei high con en in an ioxidan compounds (phenolic compounds de i ed om lignin
p ocessing) [
11
–
13
], and as a po en ial sou ce o xyloligosaccha ide p ebio ics [
14
]. They can also be
used as bio uels h ough he hyd olysis and e men a ion o cellulose o e hanol [
15
] o as a sou ce o
bioac i e polyphenols [16].
Recen ly, we ha e epo ed on ohmic hea ing, an en i onmen al iendly polyphenol ex ac ion
echnique, ha allowed an inc ease in phenolic compound ex ac ion om ine p uning esidue,
ela i e o ex ac ion a oom empe a u e [
16
]. In addi ion o que ce in, which was p esen in bo h
ex ac s, hespe idin, ellagic acid and apigenin accoun ed o he majo i y o phenolics ex ac ed by
ohmic hea ing (Table 1).
Table 1.
Majo phenolic cons i uen s o ine p uning esidue ex ac s ob ained by wo di e en
ex ac ion me hods: a oom empe a u e (RT) and by ohmic hea ing ( ine p uning esidue (VPE)).
RT VPE
Polyphenols (mg/g ex ac )
Ellagic acid
ND
2.23
Apigenin
ND
3.84
Que ce in
1.33
2.87
Hespe idin
ND
1.80
To al Polyphenols * 12 34
Fo mo e de ails see [16]. (*) Includes all compounds iden i ied [16].
Res e a ol and que ce in (and se e al o i s de i a i es) ha e been ound in many g ape ine
by-p oduc s, oge he wi h o he compounds [
5
]. Among he bioac i i ies a ibu ed o hese compounds
a e an ioxidan , an imic obial and an icance ac i i y [
16
–
18
]. Al hough posi i e heal h e ec s o o he
wine indus y esidues ha e been shown, and pha maceu ical d ugs pa en ed and ma ke ed [
19
],
ine p uning ex ac s ha e no been explo ed o his pu pose. In he p esen s udy, we apply an
en i onmen ally iendly me hod—ohmic hea ing— o ex ac ion o bioac i e compounds om ine
p uning esidue and explo e hei colon cance he apeu ic po en ial.
The an i-colon cance (an i-CRC) po en ial o polyphenol en iched ohmic ine p uning ex ac
was es ed using wo human CRC cell lines, HCT116 and RKO, wi h di e en molecula signa u es
ep esen a i e o he mos common mu a ion ypes o CRC, in o de o cha ac e ize possible molecula
a ge s and mechanisms o ac ion. Mo eo e , he majo indi idual cons i uen s p esen in he ex ac
we e es ed alone, in an a emp o iden i y possible ac i e p inciples. To iden i y possible e ec s
on sensi i i y o he chemo he apeu ical d ug 5- luo ou acil (5-FU), combina ion ea men s we e
also pe o med.
2. Ma e ials and Me hods
2.1. Reagen s
Cell cul u e media (RPMI-1640 and MEM) e al bo ine se um (FBS), an ibio ic an imyco ic solu ion,
hiazolyl blue e azolium b omide (MTT), apigenin (AP), que ce in (Q), ellagic acid (EA), hespe idin
(H), e oposide (ET), ibonuclease A (RNase A), p opidium iodide (PI), Hoechs 33342, 5- uo ou acil
(5-FU), and all eagen s no o he wise speci ied, we e pu chased om Sigma-Ald ich (S . Louis, MO,
USA). SYBR Gold nucleic acid gel s ain was acqui ed om In i ogen (Paisley, UK).
Foods 2020,9, 1102 3 o 11
2.2. Raw Ma e ial and Ex ac ion P ocess o Bioac i e Compounds
Vine p uning was ha es ed in he Po uguese egion o Minho (Ama es-B aga, Po ugal),
and samples we e d ied and c ushed as desc ibed by Jesus and collabo a o s (2019) [20].
The ex ac ion pa ame e s de ined in his s udy we e based on p e ious esul s [
16
,
20
]. B ie ly,
he ex ac ions we e ca ied ou in a glass cylind ical eac o o 30 cm o al leng h and 2.3 cm o in e nal
diame e , con aining wo s ainless s eel elec odes insula ed wi h poly e a luo oe hylene, kep a a
cons an dis ance o 7 cm. The ohmic hea ing ex ac ion o phenolic compounds ( ine p uning esidue
(VPE)) was ca ied ou wi h a liquid solid a io o 1:4, 45% hyd oalcoholic solu ion ( / ), wi h elec ical
conduc i i y adjus ed o 2.3 mS/cm wi h addi ion o NaCl ( hus gua an eeing a homogeneous cu en
low), 80
◦
C, o 60 min unde agi a ion (840.0 V/cm). The oom empe a u e ex ac (RT) was p epa ed
by placing he ine p uning inside he eac o and main aining unde cons an agi a ion by a magne ic
s i e . A he end o each ea men , he ex ac s ob ained we e immedia ely cooled in an ice ba h,
and hen il e ed wi h a cellulose il e , wi h a po e size o 0.45
µ
m. The e hanol was e apo a ed in
an o bi al shake a 40
◦
C and 150 pm, and he ex ac s lyophilized and s o ed in ambe lasks and
ozen un il use. The phenolic composi ion o he ex ac s was de e mined by Jesus and collabo a o s
(2020) [16], and he majo cons i uen s a e p esen ed in Table 1.
2.3. Cell CuL u e and Expe imen al Condi ions
HCT116 and RKO colon ca cinoma cells we e kindly p o ided by P o . Raquel Se uca (i3S/Ipa imup,
Uni e si y o Po o, Po ugal). Cells we e main ained a 37
◦
C in a humidi ied 5% CO
2
/95% ai ( / )
a mosphe e in RPMI-1640 medium con aining 10 mM HEPES, 1 mM sodium py u a e, 2.0 g/L sodium
bica bona e, 10% ( / ) FBS, and 1% ( / ) an ibio ic an imyco ic solu ion (HCT116 cells) o MEM medium
supplemen ed wi h 10 mM HEPES, 1 mM sodium py u a e, 2.2 g/L sodium bica bona e, 10% ( / ) FBS,
and 1% ( / ) an ibio ic an imyco ic (RKO cells). Cells we e seeded a 1
×
10
5
cells/mL, 1 day be o e
incuba ion wi h ex ac s/compounds.
S ock solu ions o RT, VPE, AP, Q, EA, H, ET, and 5-FU we e made in dime hyl sul oxide (DMSO),
and kep in aliquo s a
−
20
◦
C. Ex ac s/compounds we e hen added o he cul u e medium jus be o e
use, and he inal DMSO concen a ion was ne e highe han 0.5% ( / ); con ols ecei ed medium
wi h DMSO only.
2.4. Cell Viabili y
In he di e en expe imen s, e ec s on cell iabili y we e es ima ed by he MTT educ ion assay,
as p e iously desc ibed [
17
,
21
]. B ie ly, 2 h be o e he end o he ea men pe iod, MTT solu ion
was added di ec ly o each well a a inal concen a ion o 0.5 mg/mL. A e disca ding he medium,
he o mazan c ys als o med we e dissol ed wi h a 1:1 ( / ) DMSO:e hanol solu ion, and abso bance
was measu ed a 570 nm wi h backg ound sub ac ion a 690 nm. The esul s we e exp essed as
pe cen age ela i e o he con ol (cells incuba ed wi h ehicle). The concen a ion o ex ac s ha
dec eased he numbe o iable cells o 75% (IC
25
) and 50% (IC
50
) ela i e o con ol in each cell
line we e calcula ed using G aphPad P ism so wa e, e sion 7.0 (G aphPad So wa e, San Diego,
CA, USA).
2.5. DNA Damage
The abili y o he VPE and i s majo cons i uen s o induce DNA s and b eaks in HCT116 and RKO
cells a e 48 h o incuba ion was e alua ed by he alkaline e sion o he come assay, as p e iously
desc ibed [
22
,
23
]. B ie ly, cells we e collec ed by ypsiniza ion, embedded in low mel ing poin aga ose
(0.5% w/ ), and sp ead on aga ose coa ed slides. Slides we e imme sed in lysis bu e o expose
DNA, and hen in elec opho esis bu e o allow alkaline DNA unwinding. In he same solu ion,
elec opho esis was ca ied ou a 4
◦
C o 20 min a 0.8 V/cm and 300 mA. A e neu aliza ion,
slides we e s ained wi h SYBR Gold nucleic acid gel s ain, and analyzed by luo escence mic oscopy
Foods 2020,9, 1102 4 o 11
using he Come Assay IV analysis so wa e, e sion 4.3 (Pe cep i e Ins umen s L d., Su olk, UK).
A leas 100 andomly selec ed nucleoids we e sco ed pe sample and he % DNA in come ail o
ail in ensi y de e mined. Cells incuba ed wi h e oposide (10
µ
M) we e used as posi i e con ol o
DNA damage.
2.6. Cell Cycle
The e ec s o 48 h incuba ions wi h VPE o i s majo cons i uen s on cell cycle p og ession
o HCT116 and RKO cells was e alua ed by low cy ome y, as p e iously desc ibed [
24
]. B ie ly,
bo h loa ing and a ached cells we e collec ed, ixed wi h cold 70% ( / ) e hanol, washed wi h PBS,
and incuba ed o 15 min a 37
◦
C in he da k, wi h a s aining solu ion con aining 20
µ
g/mL RNase A
and 50
µ
g/mL PI. Cell cycle was analyzed using a EC800 low cy ome y analyze (Sony Bio echnology
Inc.), coun ing a leas 20,000 single cells pe sample, and he pe cen age o cells in each phase o he cell
cycle was calcula ed using he Wa son p agma ic algo i hm wi hin FlowJo analysis so wa e (FlowJo,
LLC, Ashland, OR, USA). A e sub ac ing he pe cen age o cells a he sub-G1 phase, he sum o
he h ee phases o cell cycle (G0/G1, S, and G2/M) in each es ed condi ion was equalized o 100%,
o compa e s a is ical di e ences in each phase be ween samples. E oposide (10
µ
M) was used as
posi i e con ol, as i induces G2/M cell cycle a es [25].
2.7. Cell Dea h Assessed by he Nuclea Condensa ion Assay
The e ec o VPE, AP, and H, alone o in combina ion wi h 5-FU, on HCT116 and RKO cell dea h
(48 h o p e-incuba ion ollowed by 48 h o co-incuba ion), was e alua ed by luo escence mic oscopy
assessing he p esence o nuclea condensa ion, as p e iously desc ibed [
26
]. B ie ly, bo h loa ing and
a ached cells we e collec ed, ixed wi h 4% (w/ ) pa a o maldehyde, washed wi h PBS, and a ached
o poly-L-lysine coa ed slides using a Shandon Cy ospin 4 cen i uge (The mo Scien i ic, Wal ham, MA,
USA). Then, he slides we e s ained wi h Hoechs 33342, and analyzed by luo escence mic oscopy.
A leas 500 andomly selec ed cells we e coun ed pe sample, and he pe cen age o apop o ic cells was
calcula ed om he a io be ween cells p esen ing nuclea condensa ion and he o al numbe o cells.
2.8. S a is ical Analysis
G aphPad P ism so wa e, e sion 7.0 (G aphPad So wa e, San Diego, CA, USA) was used o
g aphic ep esen a ion and s a is ical analysis o he da a. S a is ical signi icance was e alua ed by
one-o wo-way ANOVA ollowed by he Dunne ’s es , as app op ia e. Co esponding signi icance
le els we e indica ed in he igu es and espec i e legends.
3. Resul s
Vine p uning ex ac s we e p e iously ob ained by wo ex ac ion me hods— oom empe a u e
(RT) and ohmic hea ing (VPE) a 80
◦
C o 60 min—and hei phenolic compound composi ion
cha ac e ized [
16
]. He e, he ex ac s and hei majo phenolic cons i uen s we e es ed, in o de o
assess hei an i-colon cance ac i i y. The ex ac s we e es ed using wo cell lines ep esen a i e o he
main gene ic p o iles o human colon cance , HCT116 (KRAS mu a ed) and RKO (BRAF mu a ed) and
IC
50
and IC
25
alues de e mined (Table 2; Figu e S1). VPE induced highe cy o oxici y han RT, in bo h
cell lines, e lec ed in he lowe IC
50
and IC
25
alues. RKO cells we e mo e sensi i e han HCT116 o
bo h ex ac s.
The wo ex ac s ha e been chemically cha ac e ized in a p e ious s udy [
16
] and he concen a ions
o majo cons i uen s p esen in he IC
25
and IC
50
doses o VPE we e calcula ed and shown in Table 3.
The majo phenolic cons i uen s o VPE, que ce in (Q), apigenin (AP), hespe in (H), and ellagic acid
(EA) we e es ed indi idually in he concen a ions a which hey a e p esen in he IC
25
and IC
50
alues o VPE, in an a emp o iden i y he possible ac i e p inciple in he VPE ex ac wi h ega d o
an i-colon cance ac i i y.
Foods 2020,9, 1102 5 o 11
Table 2.
IC
50
and IC
25
alues o ine p uning esidue (VPR) ex ac s in HCT116 and RKO cells a e
48 h o ea men (exp essed in µg/mL).
HCT116 RKO
IC50 IC25 IC50 IC25
VPR Ex ac s (µg/mL)
RT 86.22 ±3.79 41.17 ±1.76 36.50 ±5.47 16.23 ±1.65
VPE 53.38 ±1.87 d24.41 ±1.31 d20.91 ±3.42 d4.69 ±1.16 c
Values a e exp essed as mean
±
SD o a leas h ee independen expe imen s. Le e s ep esen s a is ical signi icance:
cp≤0.001, d p≤0.0001 when compa ed wi h con ol (RT) by wo-way ANOVA.
Table 3.
Majo cons i uen s as p esen in he IC
50
and IC
25
concen a ions o he VPE ex ac in HCT116
and RKO cells (exp essed in µM).
HCT116 RKO
IC50 IC25 IC50 IC25
Majo Compounds in VPE (µM)
Apigenin (AP) 4.22 1.93 1.67 0.37
Que ce in (Q) 2.81 1.29 1.09 0.23
Ellagic acid (EA) 2.18 0.99 0.86 0.20
Hespe idin (H) 0.88 0.39 0.34 0.08
The e ec s on cell iabili y o bo h AP and Q we e simila o he e ec s o VPE in HCT116 cell cells
whe eas in RKO, H was he only compound ha alone possessed simila e ec o ha o he whole
ex ac (Figu e 1).
Foods 2020, 9, x FOR PEER REVIEW 5 o 12
IC
25
alues de e mined (Table 2; Figu e S1). VPE induced highe cy o oxici y han RT, in bo h cell lines,
e lec ed in he lowe IC
50
and IC
25
alues. RKO cells we e mo e sensi i e han HCT116 o bo h ex ac s.
Table 2. IC
50
and IC
25
alues o ine p uning esidue (VPR) ex ac s in HCT116 and RKO cells a e 48 h
o ea men (exp essed in μg/mL).
HCT116 RKO
IC
50
IC
25
IC
50
IC
25
VPR ex ac s (µg/mL)
RT 86.22 ± 3.79 41.17 ± 1.76 36.50 ± 5.47 16.23 ± 1.65
VPE 53.38 ± 1.87
d
24.41 ± 1.31
d
20.91 ± 3.42
d
4.69 ± 1.16
c
Values a e exp essed as mean ± SD o a leas h ee independen expe imen s. Le e s ep esen s a is ical
signi icance: c p ≤ 0.001, d p ≤ 0.0001 when compa ed wi h con ol (RT) by wo-way ANOVA.
The wo ex ac s ha e been chemically cha ac e ized in a p e ious s udy [16] and he concen a ions
o majo cons i uen s p esen in he IC
25
and IC
50
doses o VPE we e calcula ed and shown in Table 3. The
majo phenolic cons i uen s o VPE, que ce in (Q), apigenin (AP), hespe in (H), and ellagic acid (EA)
we e es ed indi idually in he concen a ions a which hey a e p esen in he IC
25
and IC
50
alues o
VPE, in an a emp o iden i y he possible ac i e p inciple in he VPE ex ac wi h ega d o an i-colon
cance ac i i y.
Table 3. Majo cons i uen s as p esen in he IC
50
and IC
25
concen a ions o he VPE ex ac in HCT116
and RKO cells (exp essed in μM).
HCT116 RKO
IC
50
IC
25
IC
50
IC
25
Majo compounds in VPE (µM)
Apigenin (AP) 4.22 1.93 1.67 0.37
Que ce in (Q) 2.81 1.29 1.09 0.23
Ellagic acid (EA) 2.18 0.99 0.86 0.20
Hespe idin (H) 0.88 0.39 0.34 0.08
The e ec s on cell iabili y o bo h AP and Q we e simila o he e ec s o VPE in HCT116 cell cells
whe eas in RKO, H was he only compound ha alone possessed simila e ec o ha o he whole ex ac
(Figu e 1).
Figu e 1. E ec o 48 h exposu e o VPE (IC
50
) and o i s majo cons i uen s (as p esen in espec i e IC
50
s–see
Table 3) on cell iabili y o HCT116 (A) and RKO (B) cells, as measu ed by he MTT assay. Values a e exp essed
as mean ± SEM o h ee independen expe imen s. Le e s ep esen s a is ical signi icance: a p ≤ 0.05, b p ≤ 0.01,
c p ≤ 0.001, d p ≤ 0.0001 when compa ed wi h con ol by one-way ANOVA.
Figu e 1.
E ec o 48 h exposu e o VPE (IC
50
) and o i s majo cons i uen s (as p esen in espec i e
IC
50
s–see Table 3) on cell iabili y o HCT116 (
A
) and RKO (
B
) cells, as measu ed by he MTT
assay. Values a e exp essed as mean
±
SEM o h ee independen expe imen s. Le e s ep esen
s a is ical signi icance:
a
p
≤
0.05,
b
p
≤
0.01,
c
p
≤
0.001,
d
p
≤
0.0001 when compa ed wi h con ol by
one-way ANOVA.
In o de o de e mine whe he he e ec s on cell iabili y we e due o he induc ion o DNA damage,
he come assay was used and e ec s compa ed o hose o e oposide (a known chemo he apeu ic
agen [
27
] used he e as posi i e con ol; Figu e 2and Supplemen a y Figu e S2). VPE showed o ha e
signi ican DNA damaging e ec s, al hough damage was lowe han ha induced by ET. The indi idual
compounds did no induce signi ican geno oxic e ec s (Figu e 2).
Wi h ega d o cell cycle modula ion, he posi i e con ol ET p oduced a signi ican G2/M a es in
bo h cell lines, as expec ed due o i s known geno oxic ac i i y [
27
]. VPE, on he o he hand, induced
a signi ican G0/G1 a es in bo h cell lines, and he indi idual compounds did no p oduce any
signi ican e ec ela i e o con ol cells (Figu e 3and Supplemen a y Figu e S3). These esul s a e in
ag eemen wi h he DNA damage de ec ed by he come assay.

Foods 2020,9, 1102 6 o 11
Foods 2020, 9, x FOR PEER REVIEW 6 o 12
In o de o de e mine whe he he e ec s on cell iabili y we e due o he induc ion o DNA damage,
he come assay was used and e ec s compa ed o hose o e oposide (a known chemo he apeu ic agen
[27] used he e as posi i e con ol; Figu e 2 and Supplemen a y Figu e S2). VPE showed o ha e
signi ican DNA damaging e ec s, al hough damage was lowe han ha induced by ET. The indi idual
compounds did no induce signi ican geno oxic e ec s (Figu e 2).
Figu e 2. E ec o 48 h exposu e o VPE (IC
50
) and o i s majo cons i uen s (as p esen in espec i e IC
50
s–
see Table 3) on DNA damage o HCT116 (A) and RKO (B) cells, as e alua ed by he come assay. ET (10
μ) was used as posi i e con ol. Values a e exp essed as mean ± SEM o h ee independen expe imen s.
Le e s ep esen s a is ical signi icance: b p ≤ 0.01, c p ≤ 0.001, d p ≤ 0.0001 when compa ed wi h con ol
by one-way ANOVA.
Wi h ega d o cell cycle modula ion, he posi i e con ol ET p oduced a signi ican G2/M a es in
bo h cell lines, as expec ed due o i s known geno oxic ac i i y [27]. VPE, on he o he hand, induced a
signi ican G0/G1 a es in bo h cell lines, and he indi idual compounds did no p oduce any signi ican
e ec ela i e o con ol cells (Figu e 3 and Supplemen a y Figu e S3). These esul s a e in ag eemen
wi h he DNA damage de ec ed by he come assay.
Figu e 3. E ec o 48 h exposu e o VPE (IC
50
) and o i s majo cons i uen s (as p esen in espec i e IC
50
–
see Table 3) on cell cycle o HCT116 (A) and RKO (B) cells, as assessed by low cy ome y. Values a e
exp essed as mean ± SEM o h ee independen expe imen s. Le e s ep esen s a is ical signi icance: a p
≤ 0.05, b p ≤ 0.01, c p ≤ 0.001, d p ≤ 0.0001, when compa ed wi h espec i e phase con ol by one-way
ANOVA.
When es ed in combina ion wi h 5- luo ou acil (5-FU), he pha maceu ical d ug mos equen ly
used in colon cance chemo he apy, VPE po en ia ed 5-FU e ec s a 48 h co- ea men in HCT116. AP
Figu e 2.
E ec o 48 h exposu e o VPE (IC
50
) and o i s majo cons i uen s (as p esen in espec i e
IC
50
s–see Table 3) on DNA damage o HCT116 (
A
) and RKO (
B
) cells, as e alua ed by he come assay.
ET (10
µ
) was used as posi i e con ol. Values a e exp essed as mean
±
SEM o h ee independen
expe imen s. Le e s ep esen s a is ical signi icance:
b
p
≤
0.01,
c
p
≤
0.001,
d
p
≤
0.0001 when
compa ed wi h con ol by one-way ANOVA.
Foods 2020, 9, x FOR PEER REVIEW 6 o 12
In o de o de e mine whe he he e ec s on cell iabili y we e due o he induc ion o DNA damage,
he come assay was used and e ec s compa ed o hose o e oposide (a known chemo he apeu ic agen
[27] used he e as posi i e con ol; Figu e 2 and Supplemen a y Figu e S2). VPE showed o ha e
signi ican DNA damaging e ec s, al hough damage was lowe han ha induced by ET. The indi idual
compounds did no induce signi ican geno oxic e ec s (Figu e 2).
Figu e 2. E ec o 48 h exposu e o VPE (IC
50
) and o i s majo cons i uen s (as p esen in espec i e IC
50
s–
see Table 3) on DNA damage o HCT116 (A) and RKO (B) cells, as e alua ed by he come assay. ET (10
μ) was used as posi i e con ol. Values a e exp essed as mean ± SEM o h ee independen expe imen s.
Le e s ep esen s a is ical signi icance: b p ≤ 0.01, c p ≤ 0.001, d p ≤ 0.0001 when compa ed wi h con ol
by one-way ANOVA.
Wi h ega d o cell cycle modula ion, he posi i e con ol ET p oduced a signi ican G2/M a es in
bo h cell lines, as expec ed due o i s known geno oxic ac i i y [27]. VPE, on he o he hand, induced a
signi ican G0/G1 a es in bo h cell lines, and he indi idual compounds did no p oduce any signi ican
e ec ela i e o con ol cells (Figu e 3 and Supplemen a y Figu e S3). These esul s a e in ag eemen
wi h he DNA damage de ec ed by he come assay.
Figu e 3. E ec o 48 h exposu e o VPE (IC
50
) and o i s majo cons i uen s (as p esen in espec i e IC
50
–
see Table 3) on cell cycle o HCT116 (A) and RKO (B) cells, as assessed by low cy ome y. Values a e
exp essed as mean ± SEM o h ee independen expe imen s. Le e s ep esen s a is ical signi icance: a p
≤ 0.05, b p ≤ 0.01, c p ≤ 0.001, d p ≤ 0.0001, when compa ed wi h espec i e phase con ol by one-way
ANOVA.
When es ed in combina ion wi h 5- luo ou acil (5-FU), he pha maceu ical d ug mos equen ly
used in colon cance chemo he apy, VPE po en ia ed 5-FU e ec s a 48 h co- ea men in HCT116. AP
Figu e 3.
E ec o 48 h exposu e o VPE (IC
50
) and o i s majo cons i uen s (as p esen in espec i e
IC
50
–see Table 3) on cell cycle o HCT116 (
A
) and RKO (
B
) cells, as assessed by low cy ome y.
Values a e exp essed as mean
±
SEM o h ee independen expe imen s. Le e s ep esen s a is ical
signi icance:
a
p
≤
0.05,
b
p
≤
0.01,
c
p
≤
0.001,
d
p
≤
0.0001, when compa ed wi h espec i e phase
con ol by one-way ANOVA.
When es ed in combina ion wi h 5- luo ou acil (5-FU), he pha maceu ical d ug mos equen ly
used in colon cance chemo he apy, VPE po en ia ed 5-FU e ec s a 48 h co- ea men in HCT116.
AP and H, used indi idually, we e less e ec i e. In RKO cells, he combina ion e ec s o 5-FU and VPE
we e mo e p onounced han in HCT116 a 48 h co-incuba ion. In addi ion, bo h AP and pa icula ly H
had simila e ec s o VPE in educing cell iabili y (an indi ec measu e o p oli e a ion) (Figu e 4).
Foods 2020, 9, x FOR PEER REVIEW 7 o 12
and H, used indi idually, we e less e ec i e. In RKO cells, he combina ion e ec s o 5-FU and VPE we e
mo e p onounced han in HCT116 a 48 h co-incuba ion. In addi ion, bo h AP and pa icula ly H had
simila e ec s o VPE in educing cell iabili y (an indi ec measu e o p oli e a ion) (Figu e 4).
Figu e 4. E ec o 48 h exposu e o VPE, apigenin (AP), and hespe idin (H), alone and in combina ion
wi h 5-FU on cell iabili y o HCT116 (A) and RKO (B) cells, as measu ed by he MTT assay. Cells we e
p e-incuba ed wi h he VPE (IC
25
) and co esponding concen a ions o AP, o H (see Table 3) o 48 ,
ollowed by co-incuba ion wi h 5-FU (IC
25
) o a u he 48 h (A&B). Cells ha we e no p e- ea ed we e
only exposed o 5-FU o 48 h. Values a e exp essed as mean ± SEM o h ee independen expe imen s.
Le e s and symbols ep esen s a is ical signi icance: a p ≤ 0.05,
c p ≤ 0.001, d p ≤ 0.0001 when compa ed
wi h con ol; D p ≤ 0.0001 when compa ed wi h 5-FU; ** p ≤ 0.01, *** p > 0.001, **** p ≤ 0.0001 when
compa ed wi h espec i e ex ac /compound by wo-way ANOVA.
In o de o de e mine i he po en ia ion o 5-FU by VPE sensi i i y was due o he induc ion o cell
dea h, he nuclea condensa ion assay was pe o med and he esul s a e p esen ed in Figu e 5. The da a
shows ha ela i e o 5-FU alone, he combina ion o 5-FU wi h VPE inc eased cell dea h in bo h cell
lines. In addi ion, in ag eemen wi h he p e ious esul s, RKO we e mo e sensi i e o he po en ia ion
e ec o he combina ion o 5-FU wi h VPE han HCT116. Mo eo e , in e es ing was he ac ha he
indi idual compounds did no seem o induce cell dea h, al hough hey signi ican ly dec eased cell
iabili y, pa icula ly in RKO cells.
Figu e 4.
E ec o 48 h exposu e o VPE, apigenin (AP), and hespe idin (H), alone and in combina ion
wi h 5-FU on cell iabili y o HCT116 (
A
) and RKO (
B
) cells, as measu ed by he MTT assay. Cells we e
p e-incuba ed wi h he VPE (IC
25
) and co esponding concen a ions o AP, o H (see Table 3) o 48,
ollowed by co-incuba ion wi h 5-FU (IC
25
) o a u he 48 h (
A
&
B
). Cells ha we e no p e- ea ed we e
only exposed o 5-FU o 48 h. Values a e exp essed as mean
±
SEM o h ee independen expe imen s.
Le e s and symbols ep esen s a is ical signi icance:
a
p
≤
0.05,
c
p
≤
0.001,
d
p
≤
0.0001 when compa ed
wi h con ol;
D
p
≤
0.0001 when compa ed wi h 5-FU;
** p≤0.01
,
*** p>0.001
,
**** p≤0.0001
when
compa ed wi h espec i e ex ac /compound by wo-way ANOVA.
Foods 2020,9, 1102 7 o 11
In o de o de e mine i he po en ia ion o 5-FU by VPE sensi i i y was due o he induc ion o
cell dea h, he nuclea condensa ion assay was pe o med and he esul s a e p esen ed in Figu e 5.
The da a shows ha ela i e o 5-FU alone, he combina ion o 5-FU wi h VPE inc eased cell dea h
in bo h cell lines. In addi ion, in ag eemen wi h he p e ious esul s, RKO we e mo e sensi i e o
he po en ia ion e ec o he combina ion o 5-FU wi h VPE han HCT116. Mo eo e , in e es ing was
he ac ha he indi idual compounds did no seem o induce cell dea h, al hough hey signi ican ly
dec eased cell iabili y, pa icula ly in RKO cells.
Foods 2020, 9, x FOR PEER REVIEW 7 o 12
and H, used indi idually, we e less e ec i e. In RKO cells, he combina ion e ec s o 5-FU and VPE we e
mo e p onounced han in HCT116 a 48 h co-incuba ion. In addi ion, bo h AP and pa icula ly H had
simila e ec s o VPE in educing cell iabili y (an indi ec measu e o p oli e a ion) (Figu e 4).
Figu e 4. E ec o 48 h exposu e o VPE, apigenin (AP), and hespe idin (H), alone and in combina ion
wi h 5-FU on cell iabili y o HCT116 (A) and RKO (B) cells, as measu ed by he MTT assay. Cells we e
p e-incuba ed wi h he VPE (IC
25
) and co esponding concen a ions o AP, o H (see Table 3) o 48 ,
ollowed by co-incuba ion wi h 5-FU (IC
25
) o a u he 48 h (A&B). Cells ha we e no p e- ea ed we e
only exposed o 5-FU o 48 h. Values a e exp essed as mean ± SEM o h ee independen expe imen s.
Le e s and symbols ep esen s a is ical signi icance: a p ≤ 0.05,
c p ≤ 0.001, d p ≤ 0.0001 when compa ed
wi h con ol; D p ≤ 0.0001 when compa ed wi h 5-FU; ** p ≤ 0.01, *** p > 0.001, **** p ≤ 0.0001 when
compa ed wi h espec i e ex ac /compound by wo-way ANOVA.
In o de o de e mine i he po en ia ion o 5-FU by VPE sensi i i y was due o he induc ion o cell
dea h, he nuclea condensa ion assay was pe o med and he esul s a e p esen ed in Figu e 5. The da a
shows ha ela i e o 5-FU alone, he combina ion o 5-FU wi h VPE inc eased cell dea h in bo h cell
lines. In addi ion, in ag eemen wi h he p e ious esul s, RKO we e mo e sensi i e o he po en ia ion
e ec o he combina ion o 5-FU wi h VPE han HCT116. Mo eo e , in e es ing was he ac ha he
indi idual compounds did no seem o induce cell dea h, al hough hey signi ican ly dec eased cell
iabili y, pa icula ly in RKO cells.
Figu e 5.
E ec o 48 h exposu e o VPE, apigenin (AP), and hespe idin (H), alone and in combina ion
wi h 5-FU on cell dea h o HCT116 (
A
) and RKO (
B
) cells, as e alua ed by he nuclea condensa ion
assay. Cells we e p e-incuba ed wi h he VPE (IC
25
) and co esponding concen a ions o AP, o H
(see Table 3) o 48 h, ollowed by co-incuba ion wi h 5-FU (IC
25
) o u he 48 h (
A
&
B
). Cells ha
we e no p e- ea ed we e only exposed o 5-FU o 48 h. Values a e exp essed as mean
±
SEM o h ee
independen expe imen s. Le e s and symbols ep esen s a is ical signi icance:
a
p
≤
0.05,
b
p
≤
0.01
when compa ed wi h con ol;
B
p
≤
0.01,
C
p
≤
0.001 when compa ed wi h 5-FU;
ns
p>0.05,
*
p
≤
0.05,
** p≤0.01, **** p≤0.0001 when compa ed wi h espec i e ex ac /compound by wo-way ANOVA.
4. Discussion
Ou da a show ha he ohmic hea ing ex ac ion me hod applied o ine p uning esidue was
pa icula ly e ec i e when compa ed wi h ex ac ion a oom empe a u e in ex ac ing phenolic
compounds om his wine y by-p oduc . Vine p uning esidue showed o be an excellen sou ce
o apigenin as well as o he phenolic compounds. In spi e o p e ious epo s on he p esence o
kaemp e ol, epica echin and es e a ol [
28
,
29
] in ine p uning esidues, ex ac ion o phenolic
compound om ine p uning esidue has no been su icien ly explo ed as a means o add alue o
his by-p oduc o he wine indus y. Cu en ly, his bio-was e is mos ly disposed o by bu ning in he
ields, which ep esen s a ela i ely low added alue solu ion wi h limi ed e u n.
In a p e ious s udy, we ha e shown he gene al an i-cance po en ial o ine p uning ex ac s
ob ained in di e en ways, and obse ed ha he no mal human colonocy e cell line CCD 841 CoN
was signi ican ly less sensi i e han cance cells [
16
]. Wi h ega d o an i-CRC po en ial, he e we show
ha he ex ac p oduced h ough ohmic ex ac ion o ine p uning esidue (VPE) showed be e
ac i i y han RT ex ac in bo h HCT116 and RKO cells. In addi ion, RKO cells we e mo e sensi i e
han HCT116 cells, as indica ed by he lowe IC
25
and IC
50
alues o RKO. This sugges s ha colon
umo s wi h he pa icula gene ic p o ile o RKO (BRAF mu a ed) may bene i mos om he po en ial
applica ions o VPE in an i-colon cance s a egies han KRAS mu a ed HCT116-like umo s.
The highe an i-cance ac i i y o VPE could ha e been due o he highe concen a ion o que ce in,
a compound epea edly shown o ha e an icance ac i i y (17, 18), o he p esence o ellagic acid,
hespe idin and apigenin, among o he compounds which we e absen in RT (Table 1) [
16
]. The e o e,
in an a emp o iden i y a compound ha isola ed could be esponsible o he e ec o he whole
Foods 2020,9, 1102 8 o 11
ex ac (ac i e p inciple), e ec s o he ou majo indi idual compounds we e es ed in bo h cell lines
a he concen a ions p esen in hei espec i e VPE IC
50
alues (as lis ed in Table 3), and hei e ec s
compa ed o hose o he ex ac .
In iabili y assays, AP and Q we e he mos p omising compounds in HCT116 cells, and H in
he case o RKO cells, because hey seemed o p oduce simila e ec s o hose o VPE on cell iabili y.
Que ce in, howe e , in addi ion o being p esen in bo h RT and VPE ex ac s and jus 2 imes mo e
concen a ed in VPE ela i e o RT (Table 1), seemed o ha e somewha di e en e ec s on cell iabili y
in he wo cell lines, because, while no ha ing a signi ican e ec in RKO, i signi ican ly dec eased cell
iabili y in HCT116. I seems he e o e, ha Q is no esponsible o he e ec s o VPE.
None o he e ec s o he indi idual compounds seemed o be due o DNA damage induc ion,
as demons a ed by he come assay esul s. In he come assay, he pe cen age o DNA in come ails
(% ail in ensi y) e lec s he p esence o DNA beaks. Ou da a also show he expec ed DNA damage
induc ion by he posi i e con ol e oposide (a known geno oxic agen ) (Figu e 2and Supplemen a y
Figu e S2) and, o a smalle ex en , by VPE.
DNA damage is known o induce cell cycle a es and/o cell dea h by apop osis, and u he
expe imen s we e pe o med in o de o add ess e ec s on hese pa ame e s.
Da a om cell cycle modula ion analyses e lec ed he obse ed geno oxici y o ET and VPE.
Indi idual compounds we e wi hou e ec on cell cycle egula ion in bo h cell lines, which is also in
ag eemen wi h he absence o DNA damage obse ed by he come assay (Figu e 3and Supplemen a y
Figu e S3).
In he compa ison be ween VPE and RT, VPE showed, he e o e, o be much mo e e icien han
RT agains bo h colon cance cell lines, possibly due o i s much highe phenolic con en , al hough no
indi idual majo cons i uen could be iden i ied as he ac i e p inciple. This means ha he in e es ing
an i colon cance e ec o VPE is due o he complex mix u e o compounds p esen , and no o any i s
main cons i uen s indi idually, al hough some o he indi idual compounds also showed signi ican
dec ease in cell iabili y.
Due o 5-FU’s signi icance in he ea men o CRC, combina ion assays wi h VPE and wo o he
mos e ec i e indi idual cons i uen s AP and H wi h he chemo he apeu ic d ug we e pe o med,
and e ec s on p oli e a ion and cell dea h s udied in bo h cell lines. 5-FU alone induced bo h dec eased
cell iabili y and inc eased cell dea h in bo h cell lines, as expec ed. In e es ingly, he combina ion
5-FU +VPE signi ican ly enhanced 5-FU’s e ec s on bo h pa ame e s, which indica es an inc eased cell
esponsi eness o he pha maceu ical d ug induced by VPE. Wi h ega d o he indi idual compounds,
in co- ea men wi h 5FU, AP and H had simila e ec s o VPE in RKO cells dec easing iabili y o
a simila ex en as VPE. Howe e , hose simila i ies we e no p esen when induc ion o cell dea h
was conside ed and e ec s o indi idual compounds we e also absen in HCT116 cells; VPE showed,
he e o e, o be mo e e ec i e in po en ia ing he esponse o 5-FU han any o i s main la onoids
used indi idually. This indica es ha , al hough a good sou ce o indi idual bioac i e la onoids,
in pa icula o AP, VPE as a whole would be mo e e ec i e in an i-colon cance die a y s a egies,
used, o example, in unc ionalized oods, han i s indi idual compounds.
The combina ion ea men 5-FU+VPE showed ha he e ec s in RKO we e mo e p onounced
han hose in HCT116, which is in ag eemen wi h he p e ious esponses o VPE alone. Due o he
di e en mu a ion p o iles o he cells used, and he ac ha BRAF mu a ed cells (RKO) esponded
be e o VPE han KRAS mu a ed cells (HCT116), also in combina ion ea men s wi h 5-FU, ou esul s
sugges ha pa ien s wi h colo ec al umo s ha bo ing BRAF mu a ions would bene i mos om
combina ion ea men s VPE+5-FU, compa ed wi h pa ien s wi h umo s ha bo ing KRAS mu a ions.
Al hough his is a signi ican ou come o ou s udy, u u e wo k is equi ed o con i m his end and
cla i ica ion o he molecula mechanisms in ol ed. In o de o con i m he molecula p o ile ha
would bene i he mos om VPE and VPE+5-FU, u he expe imen s should es mo e cell lines wi h
ei he BRAF o KRAS mu a ions, and quan i y exp ession le els o phospho yla ed ERK (expec ed o
be high in bo h cell lines, due o he cons i u i e ac i a ion o MAPKinase pa hway in bo h KRAS and
Foods 2020,9, 1102 9 o 11
BRAF cell lines) and AKT phospho yla ion. Since he PI3K/AKT signaling pa hway is no expec ed
o be cons i u i ely ac i a ed in BRAF mu a ed cells, bu only in KRAS mu a ed cells, de e mina ion
o VPE ea men e ec s on p-AKT le els would also be ele an o es ablish ex ac ’s e ec s on he
MAPKinase and/o he PI3K/AKT pa hways. Fu he mo e, in spi e o he ac ha ou da a sugges an
enhancemen o he e ec o he d ug 5-FU by simul aneous ea men wi h VPE, he es ablishmen o
a syne gy be ween VPE and 5-FU will equi e u he expe imen s.
Taken oge he , ou esul s show ha ohmic hea ing ex ac ion o ine p uning esidue p oduces
a polyphenol en iched ex ac (VPE) wi h an i-colo ec al cance po en ial, including he abili y o
sensi ize cells o 5-FU, a chemo he apeu ic d ug. The use o VPE in unc ional oods o nu aceu icals
could be exploi ed in pe sonalized an i colo ec al cance die a y s a egies, because highe sensi iza ion
o he d ug 5-FU is achie ed in BRAF mu a ed umo s, ela i e o KRAS mu a ed umo s. Valo iza ion
o his lignocellulosic esidue o he wine indus y should encou age bio-was e ecycling, adding alue
o his ag icul u al by-p oduc and p omo ing he sus ainable use o na u al esou ces.
Supplemen a y Ma e ials:
The ollowing a e a ailable online a h p://www.mdpi.com/2304-8158/9/8/1102/s1,
Figu e S1: E ec o VPR ex ac s on cell iabili y o HCT116 (A&B) and RKO (C&D) cells, as measu ed by he
MTT assay; Figu e S2 Rep esen a i e image ob ained by come assay showing he DNA damage caused by 48h
exposu e o e oposide (ET, posi i e con ol). Figu e S3. Rep esen a i e his og ams o he e ec o VPE (IC50) and
i s majo cons i uen s on cell cycle o HCT116 (A) and RKO (B) cells o 48 h, as assessed by low cy ome y.
Au ho Con ibu ions:
Concep ualiza ion, C.P.-W.; Fo mal Analysis, M.S.J.; In es iga ion, M.S.J. and
A.C.C.; Resou ces, J.A.T., L.D. and C.P.-W.; W i ing—O iginal D a P epa a ion, M.S.J., A.C.C. and C.P.-W.;
W i ing—Re iew and Edi ing, A.C.C. and C.P.-W.; Supe ision, C.P.-W.; Funding Acquisi ion, J.A.T., L.D. and
C.P.-W. All au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding:
This esea ch was unded by he Po uguese Founda ion o Science and Technology (FCT), unde he
scope o he s a egic unding o UIDB/04469/2020 uni and BioTecNo e ope a ion (NORTE-01-0145-FEDER-000004)
unded by he Eu opean Regional De elopmen Fund unde he scope o No e2020-P og ama Ope acional
Regional do No e. MSJ was suppo ed by he In e na ional Coope a ion P og am CNPq/CSF a he Uni e si y o
Minho, inanced by CNPq-B azilian Fede al Agency and ACC by he INTERACT p ojec –“In eg a ed Resea ch
En i onmen , Ag o-Chain and Technology”, no. NORTE-01-0145-FEDER-000017, in i s line o esea ch en i led
ISAC, co- inanced by he Eu opean Regional De elopmen Fund (ERDF), h ough NORTE 2020 (No h Regional
Ope a ional P og am 2014/2020).
Acknowledgmen s:
This s udy was suppo ed by he Po uguese Founda ion o Science and Technology (FCT),
unde he scope o he s a egic unding o UIDB/04469/2020 uni and BioTecNo e ope a ion (NORTE-01-0145-
FEDER-000004) unded by he Eu opean Regional De elopmen Fund unde he scope o No e2020-P og ama
Ope acional Regional do No e. MSJ was suppo ed by he In e na ional Coope a ion P og am CNPq/CSF a he
Uni e si y o Minho, inanced by CNPq-B azilian Fede al Agency and ACC by he INTERACT p ojec –“In eg a ed
Resea ch En i onmen , Ag o-Chain and Technology”, no. NORTE-01-0145-FEDER-000017, in i s line o esea ch
en i led ISAC, co- inanced by he Eu opean Regional De elopmen Fund (ERDF), h ough NORTE 2020 (No h
Regional Ope a ional P og am 2014/2020).
Con lic s o In e es : The au ho s decla e no con lic o in e es .
Re e ences
1.
Eu opean Commission. A Sus ainable Bioeconomy o Eu ope: S eng hening he Connec ion be ween Economy,
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