Transcript -Sitosterol activates Fas signaling in human breast cancer

Physical and mechanical properties of cardboard panels
made from used beverage carton with veneer overlay
Ayrilmis, N. ; Candan, Z. ; Hiziroglu, S. 2008. 29, 1897–1903
Student : Rattaphong Pokkaew (李平)
Student ID : 0970456
Department of Food Science
Outline
1. Introduction
2. The objective
3. Methods
4. Results
5. Conclusion
Introduction
Beverage
carton,
313000, 3%
Other
recycled
Peanut
 The peanut (Arachis hypogaea L.)
 The peanut is called as the “king” of oil seeds.
 Peanuts are also known as :
• Ground nuts, earthnuts, goobers, goober
peas,
pindas, jack nuts, pinders, manila nuts and
monkey balls.
• In the UK these are sold as monkey nuts.
Tocopherols in peanut
 Peanuts are a good source of tocopherols,
phytosterol and phospholipid
 The tocopherol content of peanuts varies
with variety and production location
 Peanut oil mainly contains -tocopherol
(50–373 ppm) and γ-tocopherols (90–390 ppm)
(Firestone, 1999)
Tocopherols in peanut
 Sturm et al. (1966) determined tocopherol content
of peanut oil from 17 varieties. Runner varieties
had higher levels of -, γ-and -tocopherols than
the Spanish varieties
 Hashim et al. (1993) reported that there were
significant differences in tocopherol content
among Runner- and Virginia-type peanut cultivars
Phospholipid in peanut
 Phospholipids (PL) contribute to the smoothness,
texture, and mouthfeel of foods
 Phospholipids improve the stability of the product
because of their inherent antioxidant properties
Breast cancer
 Breast cancer is the erratic growth
of cells that originate in the breast
tissue
 A group of rapidly dividing cells
may form a lump or mass of extra
tissue. These masses are called
tumors
 The World Health Organization
reported more than 1.2 million people
worldwide will be diagnosed with breast
cancer this year
Breast cancer
 The occurrence of breast cancer varies widely among
women from different countries and cultures
 Higher incidences in European and North American
women
 Lower in women in less developed countries and
countries relying more heavily on vegetarian diets
(American Cancer Society, 2003)
 Dietary factors, specifically the proportion of animal
versus plant fats, may play a role in the development
of breast cancer
(Messina and Barnes, 1991; Cho et al., 2003)
Breast cancer & Phytosterol
 Plant foodstuffs contain specific
phytochemicals which may offer protection
from breast cancer.
 An attractive hypothesis which may
account for the cancer protective action
of phytosterols is that phytosterols
induce apoptosis or programmed cell
death in highly proliferative tumor cells.
 Plant foodstuffs contain specific phytochemicals which may offer
protection from breast cancer.
Fig 1. Structure of some representative phytosterols
Source : Moreau et al., 2002; Berger et al., 2004; Kritchevsky and Chen, 2005
Apoptosis
 Apoptosis, or programmed cell death, is associated with several
fundamental biological processes, including cell development,
differentiation and response to injury.
 Apoptosis is defined as a set of events that once initiated induce
lethal changes that include membrane blebbing, mitochondrial
break down and DNA fragmentation
 Apoptosis occurs via two main pathways
• The intrinsic or mitochondrial pathway
• The extrinsic or death receptor-mediated pathway
Apoptotic
proteaseactivating factor1 (Apaf-1)
Cytochrome
c
Pro-apoptotic
factors
Fig 2. The intrinsic or mitochondrial pathway
Fas ligand
Fas-associated
death domain
Fas receptor
FADD
TNF
receptorassociated
death domain
Fig 3. The extrinsic or death receptor-mediated pathway
The objective
 The objective of this study was to assess the effect of cellular
supplementation with either -sitosterol or cholesterol on the
extrinsic caspase-8 pathway in the two breast cancer cell lines,
MCF-7 and MDA-MB-231.
 Hypothesized of -sitosterol, that may potentiate Fas-death
domain signaling, leading to caspase-8 activation and ultimately
to apoptosis.
 To test the hypothesis, the breast cancer cell lines were treated
with  -sitosterol or with cholesterol as a control and effects on
cell growth, sterol incorporation in cell membranes, expression
of Fas-death domain signaling proteins, and caspase-8 activity
were determined.
Methods
Cell culture
MCF-7 and MDA-MB-231 cells
1% antibiotic–antimycotic
2 g/l sodium bicarbonate
Cultured at 37 C
5% fetal bovine serum
5% CO2 / 95% air
as monolayers using RPMI 1640 growth medium
Preparation of sterol supplemented media and
measurement of cell growth
2-hydroxypropyl beta-cyclodextrin; CD complexes
(RPMI 1640 media supplemented with cholesterol or b-sitosterol)
Growth medium
(8–16 mMsterols: 5mM CD)
Control groups
-Cell were treated
with 5mM CD
Studies groups
Measurement of sterol content of cell
membranes by GLC
Cells were seeded into 6-well plates
harvested by scraping
frozen 80 C in 350 ml 10mM Tris and
20mM mannitol buffer (pH 7.4)
Samples were thawed
and briefly sonicated on ice
Samples were then
saponified at 80 C in 95% ethanolic KOH
2ml of hexane
2 ml of water
Saponified samples
The upper organic phase
Dried under nitrogen
The lower phase
GLC
The media
Determination of caspase-8 activity
were replaced
Cells were seeded in T-75 flasks;
(10,000 cells/cm2)
with sterolsupplemented
24orhcontrol
media
3 day treatment
Cells were scraped
Washed in PBS (pH 7.4)
Lysed by suspension on ice of
107 cells/200 ml in buffer
After 30 min
The lysates were aspirated
Centrifuged at 10,000g ; 4 C ; 30 min
The supernatants (cell lysates)
were analyzed by
the Bio-Rad DC protein assay
Cell lysates (100 mg
protein) were assayed
caspase-8 activity
Results
Results
Fig. 4. Effect of sterol supplementation on growth of MCF-7 cells. Cells were
grown in RPMI-1640 growth medium supplemented with different
concentrations of sterols
Results
Fig. 5. Effect of sterol supplementation on growth of MDAMB-231 cells. Cells
were grown in RPMI-1640 growth medium supplemented with different
concentrations of sterols
Table 1. Effect of 2 d-sterol supplementation on sterol content of
MCF-7 cell membranes*
Supplementation
Cholesterol
-Sitosterol
Total sterol
-Sitosterol
(mg/mg protein) (mg/mg protein) (mg/mg protein) (% total sterol)
CD vehicle
43  2a
0.0  0a
43  2a
0a
Cholesterol
49 2b
0.0  0a
49  2a
0a
-Sitosterol
40  1a
50  5b
90  5b
56b
*MCF-7
cells were treated for 2 d with 16 mM sterol or vehicle and sterol
contents of membranes determined by gas–liquid chromatography as
described. Data are means  SEM (n = 3) and letters (a, b) of values in each
column are significantly different (p<0.05).
Table 2. Effect of 2 d-sterol supplementation on sterol content of
MDA-MB-231 cell membranes*
Supplementation
Cholesterol
-Sitosterol
Total sterol
-Sitosterol
(mg/mg protein) (mg/mg protein) (mg/mg protein) (% total sterol)
CD vehicle
49  7a
0.0  0a
49  7a
0a
Cholesterol
73 4b
0.0  0a
73  4b
0a
-Sitosterol
33  2a
51  6b
84  8b
61b
*MDA-MB-213
cells were treated for 2 d with 16 mM sterol or vehicle and
sterol contents of membranes determined by gas–liquid chromatography as
described. Data are means  SEM (n = 3) and letters (a, b) of values in each
column are significantly different (p<0.05)
Table 3. Effect of 3 d-sterol supplementation on caspase-8 activity of
MCF-7 and MDA-MB-231 cell lysates*
Supplementation
CD vehicle
Caspase-8 activity of
MCF-7 lysates
6000  800a
Caspase-8 activity of
MDA-MB-231 lysates
4600  200a
Cholesterol
5000  200a
4600  200a
-Sitosterol
9800  400b
8100  800b
*Cells
were treated for 3 d with 16 mM sterol or vehicle and caspase-8
activities of cell lysates determined. Data are RFU/100 mg lysate protein and
represent means  SEM (n = 3). Letters (a, b) of values in each column are
significantly different (p<0.05).
CONT
CHOL
SIT
Fas
FasL
FADD
p-FADD
Caspase-8
Actin
Fig. 6. Effect of sterol supplementation on expression levels of Fas-related
signaling proteins in MCF-7 cells. MCF-7 cells were treated with sterols
for 24 h as described. Expression of Fas-related signaling pathway
proteins was quantified by immunoblot.
CONT
CHOL
SIT
Fas
FasL
FADD
p-FADD
Caspase-8
Actin
Fig. 7. Effect of sterol supplementation on expression levels of Fas-related
signaling proteins in MDA-MB-231 cells. MDA-MB-231 cells were treated
with sterols for 24 h as described. Expression of Fas-related signaling
proteins was quantified by immunoblot.
Conclusion
Conclusion
 -sitosterol can be affect the amounts and activity of components
of the extrinsic apoptotic pathway in human breast
adenocarcinoma cells
 -sitosterol induces a reduction in membrane sphingomyelin and
an increase the ceramide levels in some tumor cells
 The effect of -sitosterol treatment to increase caspase-8 activity
and apoptosis in these cells may be mediated, at least in part, by
changes in membrane sterol content and effects on the Fas
apoptotic pathway
THANK YOU
FOR YOUR ATTENTION !
Arachis hypogaea L
Source : http://upload.wikimedia.org/wikipedia/commons/1/1f/Koeh-163.jpg
http://en.wikipedia.org/wiki/Image:Peanuts.jpg
Preparation of sterol supplemented media and
measurement of cell growth
Studies groups
incubated for 24 h
cells were
seeded at 5000 cells/cm2 into 24-well plates
media were replaced with that containing -sitosterol or cholesterol
in graded concentrations or CD vehicle
Preparation of sterol supplemented media and
measurement of cell growth
Media were similarly changed
on days 3 and 5
Cells were trypsinized and counted on days 2, 4 and 6
by Coulter Counter using the electrical
sensing zone method
Growth curves were generated from the Coulter Counter data
buffer containing
• 10mM HEPES, pH 7.4
• 2mM EDTA
• 0.15 CHAPS
• 0.1% Triton X-100
• 5mM DTT
Fig 1. Structure of some representative phytosterols
Source : Moreau et al., 2002; Berger et al., 2004; Kritchevsky and Chen, 2005