ARTICLE IN PRESS

Phytomedicine 14 (2007) 747–754

www.elsevier.de/phymed

b-Sitosterol activates Fas signaling in human breast cancer cells

A.B. Awada,, M. Chinnama, C.S. Finka, P.G. Bradfordb
a
Department of Exercise and Nutrition Sciences and
b
Department of Pharmacology and Toxicology, State University of New York at Buffalo, Buffalo, NY 14214, USA

Abstract
b-Sitosterol is the most abundant phytosterol. Phytosterols are enriched in legumes, oil seeds and unrefined plant
oils as found in foods such as peanut butter, pistachios and sunflower seeds. b-Sitosterol inhibits the growth of several
specific types of tumor cells in vitro and decreases the size and the extent of tumor metastases in vivo. The effects of b-
sitosterol on the extrinsic apoptotic programmed cell death pathway in human breast MCF-7 and MDA-MB-231
adenocarcinoma cells were examined, along with the extent of its incorporation into cellular membranes and its effects
on cell growth, expression of Fas receptor pathway proteins, and caspase-8 activity. The results show that b-sitosterol
exposure promotes its enrichment in transformed cell membranes and significantly inhibits tumor cell growth.
Concurrently, Fas levels and caspase-8 activity are significantly increased. These actions are specific, as expression of
other proteins of the Fas receptor pathway, including Fas ligand, FADD, p-FADD and caspase-8, remain unchanged.
These findings support the hypothesis that b-sitosterol is an effective apoptosis-promoting agent and that
incorporation of more phytosterols in the diet may serve a preventive measure for breast cancer.
r 2007 Elsevier GmbH. All rights reserved.

Keywords: Phytosterols; Breast cancer; Extrinsic pathway; Apoptosis; Signal transduction

Introduction Barnes, 1991; Cho et al., 2003). Plant foodstuffs contain

specific phytochemicals which may offer protection
According to the World Health Organization, more from breast cancer. Controlled dietary studies with
than 1.2 million people worldwide will be diagnosed animals suggest that one class of phytochemicals, the
with breast cancer this year. However, the occurrence of phytosterols, may offer protection from breast cancer as
breast cancer varies widely among women from different well as protection from other cancers common to
countries and cultures, with higher incidences in Western societies, including cancers of the colon and
European and North American women as compared prostate (Awad et al., 2001, 2004; Awad and Fink,
to women in less developed countries and countries 2000). Phytosterols are cholesterol-like chemicals made
relying more heavily on vegetarian diets (American exclusively by plants. An attractive hypothesis which
Cancer Society, 2003). Dietary factors, specifically the may account for the cancer protective action of
proportion of animal versus plant fats, may play a role phytosterols is that phytosterols induce apoptosis or
in the development of breast cancer (Messina and programmed cell death in highly proliferative tumor
cells.
Corresponding author. Tel.: +1 716 829 3680x231; Apoptosis occurs via two main pathways: the intrinsic
fax: +1 716 829 3700. or mitochondrial pathway and the extrinsic or death
E-mail address: [email protected] (A.B. Awad). receptor-mediated pathway. The intrinsic pathway

0944-7113/$ - see front matter r 2007 Elsevier GmbH. All rights reserved.
doi:10.1016/j.phymed.2007.01.003
 ARTICLE IN PRESS
748 A.B. Awad et al. / Phytomedicine 14 (2007) 747–754

induces apoptosis in response to internal signals. In this USA). Antibodies, chemiluminescence luminol reagent
pathway, internal damage to the cell is sensed by the and Cruz MarkerTM molecular weight standards were
mitochondria and causes the release of apoptotic obtained from Santa Cruz Biotechnology, Inc. (Santa
protease-activating factor-1 (Apaf-1), cytochrome c, Cruz, CA, USA).
and other pro-apoptotic factors from the intermito-
chondrial membrane space (Bunz, 2001; Zörnig et al.,
2001; Saelens et al., 2004). These factors ultimately Cell culture
activate the effector protease, caspase-3, driving pro-
grammed cell death (Dragovich et al., 1998; MCF-7 and MDA-MB-231 cells, representing estro-
Fesik, 2005). The extrinsic pathway of apoptosis is gen-dependent and estrogen-independent stages of hu-
triggered by external signals that activate cell surface man breast carcinoma, were cultured at 37 1C in 5%
death receptors, including Fas. Upon binding their CO2/95% air as monolayers using RPMI 1640 growth
cognate ligands, the intracellular domains of death medium supplemented with 2 g/l sodium bicarbonate,
receptors recruit adapter molecules such as Fas-asso- 5% fetal bovine serum and 1% antibiotic–antimycotic.
ciated death domain (FADD) and TNF receptor-
associated death domain (TRADD) (Daniel et al., Preparation of sterol supplemented media and
2001), which in turn recruit procaspase-8, an initiator measurement of cell growth
caspase. Activated caspase-8 drives downstream effector
caspases, including caspase-3, ultimately culminating in RPMI 1640 media supplemented with cholesterol or
cell death. Resultant apoptotic bodies are removed by b-sitosterol as CD complexes were prepared as described
phagocytosis. (4) and added to growth medium to achieve a ratio of
The objective of this study was to assess the effect of 8–16 mM sterols: 5 mM CD. Control groups were treated
cellular supplementation with either b-sitosterol or with 5 mM CD vehicle. For growth studies, cells were
cholesterol on the extrinsic caspase-8 pathway in the seeded at 5000 cells/cm2 into 24-well plates and incu-
two breast cancer cell lines, MCF-7 and MDA-MB-231. bated for 24 h. On day 1, media were replaced with that
It is hypothesized that b-sitosterol may potentiate Fas- containing b-sitosterol or cholesterol in graded concen-
death domain signaling, leading to caspase-8 activation trations or CD vehicle. Media were similarly changed on
and ultimately to apoptosis. To test this hypothesis, the days 3 and 5. Cells were trypsinized and counted on days
breast cancer cell lines were treated with b-sitosterol or 2, 4 and 6 by Coulter Counter using the electrical
with cholesterol as a control and effects on cell growth, sensing zone method. Growth curves were generated
sterol incorporation in cell membranes, expression of from the Coulter Counter data.
Fas-death domain signaling proteins, and caspase-8
activity were determined.
Measurement of sterol content of cell membranes by
GLC
Materials and methods
Cells were seeded into 6-well plates, treated with
Materials sterols or vehicle for 2 d, and then harvested by scraping.
Cells were suspended, washed 3 times and then frozen
Breast cancer cell lines were obtained from American 80 1C in 350 ml 10 mM Tris–20 mM mannitol buffer
Type Culture Collection (Manassas, VA, USA). RPMI- (pH 7.4) until further processing. Samples were thawed
1640 media, trypsin-EDTA, antibiotic–antimycotic and and briefly sonicated on ice. An aliquot of each sample
fetal bovine serum were obtained from GibcoTM was used for protein analysis. The samples were then
Invitrogen Corporation (Grand Island, NY, USA). saponified at 80 1C in 95% ethanolic KOH in the
Cholesterol and b-sitosterol were purchased from Sigma presence of 5a-cholestane as an internal GLC standard.
Chemical Company (St. Louis, MO, USA). Protease After the addition of 2 ml of water and 2 ml of hexane
inhibitor cocktail was obtained from Roche Diagnostics the upper organic phase containing the non-saponifiable
(Mannheim, Germany). Ready gels (12% Tris–HCl, lipids (sterols) was removed and dried under nitrogen.
50 ml, 10 wells), prestained SDS-PAGE standards and The samples were reconstituted with carbon disulfide
Bio-Rad DC protein assay kit were obtained from Bio- and injected into a gas–liquid chromatograph (Shimad-
Rad Laboratories (Hercules, CA, USA). Ac-IETD-AFC zu, model GC-17A) fitted with a 30 m DB-5M5, ID
(caspase-8 substrate) and Ac-IETD-CHO (caspase-8 0.25 mm column (JRW Scientific, Folsom, CA, USA).
inhibitor) were obtained from BIOMOLs Research The column temperature was set at 265 1C and that of
Laboratories, Inc. (Plymouth Meeting, PA, USA). injection port and detector at 290 1C. Sterol contents
Cyclodextrin (2-hydroxypropyl beta-cyclodextrin; CD) were normalized to the internal standard and expressed
was obtained from Cerestar USA, Inc. (Hammond, IN, per mg protein.
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A.B. Awad et al. / Phytomedicine 14 (2007) 747–754 749

Determination of caspase-8 activity means. Data were analyzed using one-way analysis of
variance (ANOVA). Differences between mean values of
Cells were seeded in T-75 flasks at a density of the treatment groups were tested for significance
10,000 cells/cm2 and allowed to attach for 24 h at which (po0.05) using the Student’s Newman–Keuls post hoc
time the media were replaced with sterol-supplemented test. The statistical software package used was ProStat
or control media. After 3 d treatment, cells were scraped, (Poly Software Int., Pearl River, NY, USA).
washed in PBS (pH 7.4), and lysed by suspension on ice
of 107 cells/200 ml in buffer containing 10 mM HEPES,
pH 7.4, 2 mM EDTA, 0.15 CHAPS, 0.1% Triton X-100
and 5 mM DTT. After 30 min, the lysates were aspirated Results
several times through a syringe fitted with 26-guage
needle and then centrifuged at 10,000g for 30 min at b-Sitosterol supplementation inhibits MCF-7 and
4 1C. The protein concentrations of the supernatants MDA-MB-231 cell growth
(cell lysates) were analyzed by the Bio-Rad DC protein
assay modified for thiol containing samples. The effects of supplementation with cholesterol or b-
To quantify caspase-8 activity, cell lysates (100 mg sitosterol on cell growth were studied with the breast
protein) were assayed with the specific caspase-8 cancer cell lines MCF-7 and MDA-MB-231. For MCF-
substrate, Ac-IETD-AFC, in the presence and absence 7 cells, cholesterol supplementation (8 and 16 mM)
of the specific caspase-8 inhibitor, Ac-IETD-CHO. resulted in significantly increased cell growth when
Samples were incubated with 50 mM Ac-IETD-CHO compared to vehicle control after 3 and 5 d supplemen-
for 30 min at 37 1C. This inhibitor blank was used to tation (Fig. 1). There was a dose-dependency of the
correct each sample for its own respective caspase-8 effect, with 8 and 16 mM cholesterol significantly
activity. Ac-IETD-AFC was then added to 50 mM and increasing cell growth by 29% and 60%, respectively.
the samples incubated at 37 1C. Relative fluorescence In contrast, supplementation with 8 or 16 mM b-
was monitored over time (1, 2, 3, 4, 19, and 20 h) at an sitosterol for 1, 3, and 5 d resulted in significantly
excitation wavelength of 360 nm and an emission decreased cell growth when compared to vehicle control.
wavelength of 530 nm using a fluorescence multi-well At 5 d, the effect of 16 mM b-sitosterol (81% decrease)
plate reader (CytoFluorTM II, PerSeptive Biosystems). was greater than that of 8 mM b-sitosterol (63%
Fluorescence values obtained for samples treated with decrease) after 5 d supplementation.
the specific caspase-8 inhibitor were subtracted from The effect of sterol supplementation on the cell
those that did not receive inhibitor. growth of MDA-MB-231 cells was also examined and

300
Western immunoblot analysis Control
Cell Density (cell # x 103/cm2)

Chol 8 uM a
250 Chol 16 uM a

Western immunoblot analyses for Fas, FasL, FADD, Sit 8 uM

Sit 16 uM
b b

p-FADD, caspase 8 and b-actin were performed on cell 200

lysates (50 mg protein) resolved by SDS-PAGE (12% 150

c
c
polyacrylamide gels) and electroblotted onto PVDF a
membranes. After blocking with 5% nonfat dried milk 100
a
and 0.05% Tween20 Tris-buffered saline (TBS-T), blots b
d
d

were incubated for 1 h with primary antibody, washed 3 50 e

c d
times with TBS-T and incubated with the secondary 0
antibody. Blots were treated with luminol reagent for 1 3 5
1 min and exposed to X-ray film. Intensities were Days of Treatment

analyzed by densitometry (Bio-Rad Imaging Densit- Fig. 1. Effect of sterol supplementation on growth of MCF-7
ometer, Model GS-700) and quantified using Quantity cells. Cells were grown in RPMI-1640 growth medium
One (Version 4.2.3) software. Blots were stripped and supplemented with different concentrations of sterols: 8 or
re-probed for b-actin reactivity. Experimental values 16 mM cholesterol; 8 or 16 mM b-sitosterol. Control group
were normalized to b-actin reactivity. received only 5 mM of the cyclodextrin vehicle. Cell growth, as
assessed by counter coulter, was studied up to 5 d sterol
supplementation. Values (mean7SEM, n ¼ 3) with different
letters (a–e) in one time point are significantly different
Statistical analyses (po0.05) [Control: 5 mM cyclodextrin (CD) vehicle; Chol
8 mM: 8 mM cholesterol in CD; Chol 16 mM: 16 mM cholesterol
All experiments were performed in triplicate. Values in CD; Sit 8 mM: 8 mM b-sitosterol in CD; Sit 16 mM: 16 mM b-
are expressed as means and standard errors of the sitosterol in CD].
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750 A.B. Awad et al. / Phytomedicine 14 (2007) 747–754

observed to be similar to the effect on MCF-7 cells with either 8 or 16 mM b-sitosterol significantly de-
(Fig. 2). Cholesterol supplementation at 8 and 16 mM for creased cell growth compared to vehicle control after 1,
5 d resulted in significantly increased cell growth 3 and 5 d sterol supplementation. However, there was no
compared to vehicle control. After 3 d, the effect of significant difference between the two b-sitosterol
16 mM cholesterol was greater than that of 8 mM concentrations on any of the days studied.
cholesterol; however, there was no difference in cell
growth between the two cholesterol concentrations after
5 d sterol supplementation. Like the results seen with Sterol supplementation alters composition of MCF-7
MCF-7 cells, supplementation of MDA-MB-231 cells and MDA-MB-231 cell membranes

450
Supplementation of MCF-7 cells with 16 mM choles-
Control a
Chol 8 uM terol resulted in significantly increased cholesterol
400
Cell Density (cell # x 103/cm2)

Chol 16 uM a content of the crude membrane preparations compared

350 Sit 8 uM
Sit 16 uM a b to CD vehicle control or b-sitosterol groups (Table 1).
300 Likewise, supplementation of MCF-7 cells with 16 mM
250 b b-sitosterol significantly increased the b-sitosterol con-
200 tent of crude cell membranes. In the control and
150
c cholesterol supplementation groups, no detectable levels
a c of b-sitosterol were observed. However, total sterol
100
a
a d content of membranes from b-sitosterol-supplemented
50 b c
b d
MCF-7 cells was significantly greater and about twice
0 that of the CD control and cholesterol groups, reflecting
1 3 5
Days of Treatment the enrichment of membranes with b-sitosterol.
With MDA-MB-231 cells, cholesterol supplementa-
Fig. 2. Effect of sterol supplementation on growth of MDA- tion resulted in significant elevation of total membrane
MB-231 cells. Cells were grown in RPMI-1640 growth medium sterol content compared to the CD vehicle control or b-
supplemented with different concentrations of sterols: 8 or
sitosterol groups (Table 2). Likewise, when treated with
16 mM cholesterol; 8 or 16 mM b-sitosterol. Control group
b-sitosterol, MDA-MB-231 cell membranes contained
received only 5 mM of cyclodextrin vehicle. Cell growth, as
assessed by counter coulter, was studied up to 5 d sterol significantly greater amounts of b-sitosterol when
supplementation. Values (mean7SEM, n ¼ 3) with different compared to control or cholesterol-treated groups. b-
letters (a–d) are significantly different (po0.05) [Control: Sitosterol was not detected in either the control or
5 mM cyclodextrin (CD) vehicle; Chol 8 mM: 8 mM cholesterol cholesterol treatment group. b-Sitosterol constituted
in CD; Chol 16 mM: 16 mM cholesterol in CD; Sit 8 mM: 8 mM 61% of the total sterol content of crude cell membranes
b-sitosterol in CD; Sit 16 mM: 16 mM b-sitosterol in CD]. of MDA-MB-231 cells after supplementation with b-

Table 1. Effect of 2 d-sterol supplementation on sterol content of MCF-7 cell membranes

Supplementation Cholesterol (mg/mg b-Sitosterol (mg/mg Total sterol (mg/mg b-Sitosterol (% total
protein) protein) protein) sterol)

CD vehicle 4372a 0.070a 4372a 0a

Cholesterol 4972b 0.070a 4972a 0a
b-Sitosterol 4071a 5075b 9075b 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 means7SEM (n ¼ 3) and letters (a, b) of values in each column are significantly different (po0.05).

Table 2. Effect of 2 d-sterol supplementation on sterol content of MDA-MB-231 cell membranes

Supplementation Cholesterol (mg/mg b-Sitosterol (mg/mg Total sterol (mg/mg b-Sitosterol (% total
protein) protein) protein) sterol)

CD vehicle 4977a 0.070a 4977a 0a

Cholesterol 7374b 0.070a 7374b 0a
b-Sitosterol 3372a 5176b 8478b 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 means7SEM (n ¼ 3) and letters (a, b) of values in each column are significantly different (po0.05).
 ARTICLE IN PRESS
A.B. Awad et al. / Phytomedicine 14 (2007) 747–754 751

sitosterol. The total membrane sterol levels of the compared to the CD vehicle control group (1.9-fold
cholesterol- and b-sitosterol-supplemented groups were increase) or the cholesterol treatment group (2.9-fold
similar to each other and both significantly greater than increase) (Table 3). There was no significant difference
the CD control group. in caspase-8 activity between the CD vehicle control and
cholesterol groups at any time point. Similarly, in b-
sitosterol-supplemented MDA-MB-231 cells, caspase-8
b-Sitosterol increases caspase-8 activity in MCF-7 activity was significantly increased when compared to
and MDA-MB-231 cells the CD vehicle control or cholesterol treatment groups
(Table 3). There was no significant difference in caspase-
Caspase-8 activities in lysates from 3 d sterol-supple- 8 activity between the vehicle control and the cholesterol
mented MCF-7 and MDA-MB-231 cells were measured treated groups.
kinetically over 1–20 h of incubation with the caspase-8
substrate, Ac-IETD-AFC. Activities increased linearly
and in parallel for the 20 h incubation period in all the Fas expression is selectively induced by b-sitosterol
treatment groups and results for only the 20 h time
points are reported. In b-sitosterol-supplemented MCF- The expression levels of the Fas-related proteins Fas,
7 cells, caspase-8 activity was significantly increased FasL, FADD, phosphorylated FADD (p-FADD), and
caspase 8 were assessed by quantitative immunoblot.
After 6 h of sterol supplementation of MCF-7 cells,
Table 3. Effect of 3 d-sterol supplementation on caspase-8 there were no detectable differences in the amounts of
activity of MCF-7 and MDA-MB-231 cell lysates Fas, FasL, FADD, p-FADD and caspase 8 between the
two sterol treatment groups (data not shown). However,
Supplementation Caspase-8 Caspase-8 activity of after 24 h b-sitosterol supplementation of MCF-7 cells,
activity of MCF- MDA-MB-231
there was a 30% increase in Fas protein level compared
7 lysates lysates
to MCF-7 cells supplemented with cholesterol or CD
CD vehicle 60007800a 46007200a vehicle control (Fig. 3). No differences in any other Fas-
Cholesterol 50007200a 46007200a related proteins were observed at either time among the
b-Sitosterol 98007400b 81007800b three supplementation groups. Similar data were ob-
 Cells were treated for 3 d with 16 mM sterol or vehicle and caspase- tained from sterol-supplemented MDA-MB-231 cells;
8 activities of cell lysates determined. Data are RFU/100 mg lysate however, the b-sitosterol dependent increase in Fas
protein and represent means7SEM (n ¼ 3). Letters (a, b) of values in protein levels, though significant, was only 10% above
each column are significantly different (po0.05). control after 24 h (Fig. 4).

CONT CHOL SIT

Fas

FasL

FADD

p-FADD

Caspase-8

Actin

Fig. 3. 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.
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752 A.B. Awad et al. / Phytomedicine 14 (2007) 747–754

CONT CHOL SIT

Fas

FasL

FADD

p-FADD

Caspase-8

Actin

Fig. 4. 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.

Discussion growth of several specific cancer cell lines representing

common cancers of Western society. The present
Preliminary studies showed that phytosterol supple- findings are contrary to a previous report (Ju et al.,
mentation of MDA-MB-231 human breast cancer cells 2004) in which treatment of MCF-7 cells with b-
increases the activities of caspases-3, 8, and 9 (Awad et sitosterol at concentrations of 150 mM increased cell
al., 2003). The purpose of the present study was to growth. Several reasons could account for this discre-
expand this work by examining the effects of b-sitosterol pancy, perhaps most significant is that b-sitosterol is not
on caspase activity in MCF-7 human breast cancer cells readily soluble at concentrations in the hundred micro-
as well as to determine the effects of b-sitosterol on cell molar range.
growth, membrane sterol content, and expression levels In addition to the negative effect of b-sitosterol on cell
of Fas-related apoptotic proteins in both cell lines. The growth, b-sitosterol treatment of tumor cells is asso-
MCF-7 cell line is estrogen receptor-positive and ciated with increased apoptosis (Awad et al., 2004). In
considered a model of early stage breast cancer whereas the present study, potential cellular mechanisms under-
the MDA-MB-231 cell line is estrogen receptor-nega- lying phytosterol-induced apoptosis were investigated.
tive, hormone-insensitive, and a model of late or Specific effects on caspase-8, the initiator caspase in the
advanced stage breast cancer. The effect of sterols on extrinsic apoptotic pathway, were identified. After 3 d,
the cell growth and the expression of the extrinsic b-sitosterol induced a 1.9-fold increase in caspase-8
apoptotic pathway in these cells have not been studied. activity in MCF-7 cells and a 2.9-fold increase in MDA-
b-Sitosterol was observed to have growth inhibitory MB-231 cells. These effects were specific and not
effects on both breast cancer cell lines and these effects mimicked by cholesterol.
occurred over similar time (3–5 d) and concentration b-Sitosterol is a plant sterol similar in structure to
ranges (8–16 mM) in both cell types. The growth cholesterol. Supplementation of cells with b-sitosterol
inhibitory effect of phytosterols is at concentrations was hypothesized to lead to its incorporation into
relevant to vegetarian diets similar to that observed with cellular membranes. This action might affect signal
other tumor cell lines, including the human prostate transduction pathways leading to cell death. Treatment
cancer LNCaP cell line (von Holtz et al., 1998) and the of MCF-7 and MDA-MB-231 cells with b-sitosterol and
human colon cancer HT-29 cell line (Awad et al., 1998). cholesterol resulted in significant increases in the
These findings indicate that b-sitosterol inhibits the amounts of sterol incorporation in cellular membranes.
 ARTICLE IN PRESS
A.B. Awad et al. / Phytomedicine 14 (2007) 747–754 753

b-Sitosterol treatment of MCF-7 cells did not lower the be a consequence of enrichment of b-sitosterol into
membrane cholesterol content when compared with the cellular membranes. Similar to MCF-7 cells, treatment
control group but rather enriched the cellular mem- of MDA-MB-231 cells with b-sitosterol for 24 h did not
branes with b-sitosterol, which under the specific show any detectable changes in the levels of other
incubation conditions, resulted in b-sitosterol represent- proteins of the extrinsic apoptotic pathway such as
ing more than half of the total membrane sterols. The FasL, FADD, p-FADD and caspase-8 when compared
total sterol content of membranes from b-sitosterol with the control and cholesterol treatment groups.
treatment group was about two-fold that of the control In summary, the present study demonstrates that
or cholesterol treatment groups. The cholesterol-treat- membrane enrichment with the phytosterol, b-sitosterol,
ment control produced a 14% increase in cholesterol may affect the amounts and activity of components of
content of MCF-7 membranes when compared with the the extrinsic apoptotic pathway in human breast
vehicle control. Similar results were obtained in MDA- adenocarcinoma cells. This is a significant observation
MB-231 cells, wherein b-sitosterol constituted 61% of and investigation of any cause and effect relationship
total sterols. Treatment of MDA-MB-231 cells with between these observations is warranted. It is interesting
cholesterol however resulted in about 49% increase in to consider that the Fas receptor has been reported to be
cholesterol content of cell membranes. The total sterol localized in cholesterol- and sphingomyelin-rich mem-
contents in cholesterol and b-sitosterol treatment groups brane rafts (Hueber et al., 2002). Perhaps any disruption
were similar and both were significantly greater than or change in the cholesterol–sphingomyelin ratio or
that of the control group in this cell line. This sterol content by the addition of phytosterols may
enrichment of membranes with sterols could influence influence the activities of lipid rafts and thus expression
the cell signaling mechanisms. of and signaling through Fas receptors. Our previous
These observations led to the suggestion that the Fas observations indicate that b-sitosterol induces a reduc-
receptor-mediated signal transduction pathway leading tion in membrane sphingomyelin and an increase the
to activation of caspase-8 might be influenced by ceramide levels in some tumor cells (von Holtz et al.,
phytosterol-induced changes in cellular membranes. 1998; Awad et al., 1998). Ceramide could play a role in
Western blot evaluation of a series of Fas-related the activation of the extrinsic pathway as suggested by
apoptotic proteins in b-sitosterol-treated cells showed observations of death receptor clustering in ceramide-
no effects on the expression levels of FasL, FADD, p- rich lipid rafts (Hueber, 2003; Scheel-Toellner et al.,
FADD and caspase-8. However, there was a 30% 2004). These findings suggest that the effect of b-
increase in Fas expression in MCF-7 cells after 24 h b- sitosterol treatment to increase caspase-8 activity and
sitosterol treatment. This finding suggests a specific apoptosis in these cells may be mediated, at least in part,
effect of b-sitosterol supplementation on Fas expression by changes in membrane sterol content and effects on
and that this increase might be responsible for the the Fas apoptotic pathway.
increased activity (1.9-fold) of the initiator caspase,
caspase-8.
The increased expression of Fas, a transmembrane
protein, upon treatment with b-sitosterol may be related Acknowledgment
to the enrichment of cellular membranes with b-
sitosterol. The kinetics of these events deserves con- The support of The Peanut Institute for this project is
sideration. After 6 h treatment (data not shown) of appreciated.
MCF-7 cells with b-sitosterol there were no detectable
differences in expression of Fas, FasL, FADD, p-
FADD and caspase-8 across the treatment groups. References
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