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ANTICARCINOGENIC EFFECT OF PHYTOSTEROLS AND/OR ANTICARCINOGENIC EFFECT OF PHYTOSTEROLS AND/OR
BETABETA--CRYPTOXANTHIN IN COLON CANCER CELLSCRYPTOXANTHIN IN COLON CANCER CELLS
I
Nutrition and Food Science Area.
Faculty of Pharmacy. University of Valencia
Antonio Cilla Tatay PhDPostdoctoral researcher
E-mail: antonio.cilla@uv.es
BACKGROUND
DETERMINANTS OF HEALTH
Lalonde (1974)
BACKGROUND
Bygbjerg, I.C. Science (2012) 337: 1499-1501 Yach et al. JAMA (2004) 291: 2616-2622
BACKGROUND
Colorectal cancer incidence rates by sex
Developed countries Developing countries
Center et al. CA Cancer J Clin (2009)59:366–378
BACKGROUND
Aetiology of colon cancer
� Colorectal cancer is widely believed tobe an enviromental disease
� Heredity
Culture
Social status
Lifestyle practices Dietary habits
ALCOHOL
SMOKING
HYPERCALORIC FOODSCHEMICALS
HEALTHY DIET PHYSICAL ACTIVITY
BACKGROUND
Steps and evolution of colon cancer Chemoprevention by phytochemicals
Normal
epithelium
Early adenoma
Intermediate
adenoma
Carcinoma
Late adenoma
Transformation
(initiation)
Promotion (tumoral
growing)
Invasion
(distant metastasis)
Manson, M.M. Trends Mol. Med. (2003) 9: 11-18
BACKGROUND
Functional food trends in 2014
http://www.foodmag.com.au/news/naturally-functional-foods-to-lead-the-way-in-2014
BACKGROUND
Functional foods
Classical Fiber & carbohydrates Bioactive
Vitamins Cereals PhytosterolsVitamins
Minerals
Aminoacids
Probiotics
Cereals
β-glucans
Soy
Resistant starch
Inulin
Fruit fiber
Phytosterols
Carotenoids
Polyphenols
Antioxidant extracts(cocoa, grape seed)
ω-3 and ω-6
β-sitosterol , campesterol and stigmasterol
β-cryptoxanthin
Saura-Calixto et al., (2005)
BACKGROUND
WHY THESE COMPOUNDS?
Gastrointestinal and systemic effects of a doubly modified functional beverage containing
β-cryptoxanthin and phytosterols; unravelling potential mechanisms and mediators.
National Research Project (AGL2012-39503-C02-01)
β-cryptoxanthin beneficial properties in different bone remodeling markers (formation and resorption)
Phytosterols hypocholesterolemic effect
Combined action against colon cancer?
BACKGROUND
Proposed mechanisms of action of phytosterols and carotenoids on carcinogenesis
Tanaka et al. Molecules (2012) 17: 3202-3242Woyengo, et al. Eur. J. Clin. Nutr. (2009) 63: 813-820
OBJECTIVES
1. Evaluate the antiproliferative activity of maindietary phytosterols and/or β-cryptoxanthin against
a colon cancer cell line (Caco-2 cells).
2. Unravel the biochemical and molecular2. Unravel the biochemical and molecularmechanisms involved in their possible
antiproliferative activity.
SAMPLES AND METHODS
SAMPLES
Standards (alone or in combination) at phyisiological human serum
concentrations according to:
• Cilla et al., Ann Nutr. Metab. (2011) 58, suppl 3, 104
• Granado-Lorencio et al., J. Agric. Food Chem. (2011) 59, 11819-24
METHODS
Cell treatment
(continuous incubation24 h with
phytochemicalstandards)
β-cryptoxanthin β-sitosterol
Campesterol Stigmasterol
Caco-2 cells
standards)
• Cell viability - MTT test (Spectrophotometry)
• Cell cycle (Flow cytometry)
• Apoptosis Annexin V (Flow cytometry)
• Mitochondrial membrane potential (Flow cytometry)
• Intracellular Ca2+ (Flow cytometry)
• Reactive oxygen species ROS (Flow cytometry)
• Total thiols (Spectrophotometry)
• Pro-apoptotic BAD protein (Western blot)
• PARP-1 / caspase-3 susbtrate (Western blot)
RESULTS
60
80
100
120
(% d
el co
ntrol)
MTT test a
cdefg efg
cdefgcdefg
ab
a
cdef
bc
g
defg cdefg
g fg
efg
cde
cdefgcdef
bcd
A) IMPACT ON VIABILITY AND APOPTOSIS
Different letters denote statistical significant differences (p<0.05) using one-way ANOVA followed by LSD post-hoc test.
0
20
40
60
Via
bility
(% d
el co
ntrol)
RESULTS
A) IMPACT ON VIABILITY AND APOPTOSIS
50
60
70
(%)
Cell cycle distribution
Sub-G1
G0/G1
S
G2/M
Sub-G1
G0/G1
S
G2/M
An asterisk indicates statistically significant diferences (p<0.05) versus control using one-way ANOVA followed by LSD post-hoc test
0
10
20
30
40
50
Control β-CX (3 µM) β-sit (12 µM) Camp (1 µM) Stig (0.25 µM) PS mix β-CX + PS mix
Cell
phase
dis
trib
ution
(%)
Sub-G1
G1
S
G2/M
*
*
** *
*
RESULTS
A) IMPACT ON VIABILITY AND APOPTOSIS
Control
β-sit (12µM)β-CX (3 µM)
PI
PI
Annexin V Annexin V
Apoptosis (Annexin V)
Control
β-CX+PS mixPS mix
Stig (0.25 µM)Camp (1 µM)
PI
PI
PI
PI PI
Annexin V
Annexin V
Annexin V Annexin V
Annexin V Annexin V
RESULTS
A) IMPACT ON VIABILITY AND APOPTOSIS
100
120
mem
bra
ne p
ote
ntial (%
of
control)
*
Changes in mitochondrial membrane potential (MMP)
An asterisk indicates statistically significant diferences (p<0.05) versus control using one-way ANOVA followed by LSD post-hoc test
0
20
40
60
80
Control β-Cx (3 µM) β-sit (12 µM) Camp (1 µM) Stig (0.25 µM) PS mix β-Cx + PS mix
Mitoch
ondrial m
em
bra
ne p
ote
ntial (%
of
control)
**
* * *
RESULTS
A) IMPACT ON VIABILITY AND APOPTOSIS
Stig βCampβ-CX β-sit2.1
Poly(ADP-ribose)polymerase (PARP) cleavage
ControlStig
(0.25 µM) PS mixβ-CX + PS
mix
Camp
(1 µM)
β-CX
(3 µM)β-sit
(12 µM)
PARP (116KDa)
Cleaved product (89KDa)
Actin
0
0.7
1.4
2.1
PA
RP
11
6:a
ctin
rat
io
(arb
itra
ry u
nit
s)
a
b
cd dd
b
RESULTS
B) REDOX STATE
Control Stig (0.25 µM)Camp (1 µM)
β-sit (12µM)β-CX (3 µM)
Reactive oxygen species
(ROS)
Control
β-CX+PS mixPS mix
Stig (0.25 µM)Camp (1 µM)
RESULTS
B) REDOX STATE
120
140
160
180
Tota
l th
iols
(%
of
control)
a
bc
cd
b
cd
e
de
Total thiols (-SH)
Different letters (a-e) denote statistical significant differences (p<0.05) using one-way ANOVA followed by LSD post-hoc test.
0
20
40
60
80
100
120
Control β-CX (3 µM) β-Sit (12 µM) Camp (1 µM) Stig (0.25 µM) PS mix β-CX + PS mix
Tota
l th
iols
(%
of
control)
a
RESULTS
C) Ca2+ AND RELATED SIGNALLING PATHWAYS
Cytosolic calcium influx
25
30
35
% intrace
llula
r ca
lciu
m
e
Different letters (a-e) indicate statistically significant diferences (p<0.05) versus control using one-way ANOVA followed by LSD post-hoc test
0
5
10
15
20
Control β-CX (3 µM) β-sit (12 µM) Camp (1 µM) Stig (0.25 µM) PS mix β-CX + PS mix
% intrace
llula
r ca
lciu
m
bc
bccd
d
a
ab
RESULTS
C) Ca2+ AND RELATED SIGNALLING PATHWAYS
Pro-apoptotic BAD dephosphorilation
ControlStig
(0.25 µM) PS mixβ-CX + PS
mix
Camp
(1 µM)
β-CX
(3 µM)
β-sit
(12 µM)
p-BAD
(Ser 136)
Actin
0
1.1
2.2
BA
D-P
se
r13
6:a
ctin
(arb
itra
ry u
nit
s)
a
bc c
dbc
d
RESULTS
β-cryptoxanthin / β-sitosterolCampesterol / Stigmasterol
OVERVIEW
Adapted from Vejux et al., Braz. J. Med. Biol. Res. (2008) 41, 545-556
ExternalizationExternalization of of phosphatidylserinephosphatidylserineRiseRise in in cellcell cyclecycle subsub--G1G1
CONCLUSIONS
� β-cryptoxanthin and/or main dietary phytosterols (alone and
in combination) reduced cell growth of colon cancer Caco-2
cells up to 44% due to apoptosis, possibly through the
mitochondrial pathway
� No clear dose-response was observed, neither additive nor� No clear dose-response was observed, neither additive nor
synergistic effect for mixtures, but they retain the same
antiproliferative activity as individual compounds indicating
absence of antagonistic actions
� The effects were obtained with concentrations compatible
with physiological serum levels in humans and with reported
bioavailability of these phytochemicals after regular
consumption of a beverage containing a mix of all these
molecules
FUTURE RESEARCH
Functional beverages
containing phytosterols and
β-cryptoxanthin
Colon cancer (Caco-2 cells)
• In vitro gastrointestinal digestion• In vitro colonic fermentation
Anti-proliferative activity
ACKNOWLEDGEMENTS
Laboratory of Biochemistry
(STEBICEF).
University of Palermo
� Dr. Maria Antonia Livrea
� Dr. Luisa Tesoriere
� Dr. Alessandro Attanzio
Bionutest research group.
University of Valencia
� Dr. Reyes Barberá
� Dr. María Jesús Lagarda� Dr. Amparo Alegría� Dr. Guadalupe García- Llatas� Lorena Claumarchirant PhD student
� Dr. Alessandro Attanzio
� Dr. Mario Allegra
Thank you for your attention
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