Differential effects of cholesterol and phytosterols on cell proliferation, apoptosis and expression of a prostate specific gene in prostate cancer cell lines

Differential effects of cholesterol and phytosterols on cell

proliferation, apoptosis and expression of a prostate

specific gene in prostate cancer cell lines

Godwin O. Ifere PhDa,*, Erika Barr PhDa, Anita Equan MDa,Kereen Gordon MSca, Udai P. Singh PhDb, Jaideep Chaudhary PhDa,

Joseph U. Igietseme PhDc, Godwin A. Ananaba PhDa

a Department of Biological Sciences, and Center for Cancer Research and Therapeutic Development,

Clark Atlanta University, Atlanta, GA 30314, USAb Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, Atlanta, GA 30310, USA

c Molecular Pathogenesis Lab, National Center for Infectious Diseases, Centers for Disease Control & Prevention (CDC),

Atlanta, GA 30333, USA

Accepted 20 December 2008

Abstract

Background: The purpose of our study was to show the apoptotic and anti-proliferative effects of phytosterols as distinct from cholesterol

effects on prostate cancer cell lines, and also their differential expression of caveolin-1, and a prostate specific gene, PCGEM1. Methods: PC-3

and DU145 cells were treated with sterols (cholesterol and phytosterols) for 48 h, followed by trypan blue dye exclusion measurement of

cytotoxicity and MTT cell proliferation assays, respectively. Cell cycle analysis was carried out microscopically, and by propidium iodide

uptake using flow cytometry. Sterol induction of oncogenic gene expression was evaluated by RT-PCR. Apoptotic cells were identified by

immunocytochemistry using DNA fragmentation method, and by annexin V adhesion using flow cytometry. Results: Physiological doses

(16 mM) of these sterols were not cytotoxic in these cells. Cholesterol-enrichment promoted mitosis (54 and 61% by microscopy; 40.8 and

34.08% by FACS analysis in PC-3 and DU145, respectively) and cell growth (P < 0.05), while phytosterols suppressed mitosis (29 and 35%

by microscopy; 27.71 and 17.37% by FACS analysis in PC-3 and DU145, respectively), and significantly induced tumor-suppression

(P < 0.05) and apoptosis. We demonstrated for the first time that cholesterols upregulated the expression of PCGEM1 even in androgen-

insensitive prostate cancer cell lines. Phytosterols reversed this effect, while upregulating the expression of caveolin-1, a known mediator of

androgen-dependent proto-oncogene signals that presumably control growth and anti-apoptosis. Conclusions: Phytosterol inhibition of

PCGEM1 and cell growth and the overexpression of caveolin-1, suggests that poor disease prognosis anchors on the ability of caveolin-1 to

regulate downstream oncogene(s) and apoptosis genes. Sterol intake may contribute to the disparity in incidence of prostate cancer, and

elucidation of the mechanism for modulation of growth and apoptosis signaling may reveal potential targets for cancer prevention and/or

chemotherapeutic intervention. Sterol regulation of PCGEM1 expression suggests its potential as biomarker for prediction of neoplasms that

would be responsive to chemoprevention by phytosterols.

Published by Elsevier Ltd.

Keywords: Prostate cancer; Caveolin-1; Sterols; Cholesterol; Phytosterols; PCGEM1; Apoptosis; Proto-oncogene; Biomarkers; Chemoprevention

www.elsevier.com/locate/cdp

Cancer Detection and Prevention 32 (2009) 319–328

* Corresponding author at: Department of Biological Sciences, Clark

Atlanta University, 223 James P. Brawley DR. S.W., Atlanta, GA 30314,

USA. Tel.: +1 404 880 6977; fax: +1 404 880 8065.

E-mail address: [email protected] (G.O. Ifere).

0361-090X/$ – see front matter. Published by Elsevier Ltd.

doi:10.1016/j.cdp.2008.12.002

1. Introduction

High prevalence and poor prognosis of prostate cancer has

been associated with increased intake of saturated fats and

cholesterol [1,2]. To the contrary, dietary phytosterols, which

are plant cholesterol counterparts, have reportedly suppres-

G.O. Ifere et al. / Cancer Detection and Prevention 32 (2009) 319–328320

sive effects on cell growth [3,4]. The contribution of high

sterol diets to the etiology, progression or prevention/reversal

of prostate cancer may be understood from cholesterol’s

crucial role in membrane organization, dynamics, function

and sorting [5]. Associated with membrane cholesterol is the

protein caveolin-1 (cav-1), a vital component of caveolae,

which are cytomorphologically pitted vesicular membrane

invaginations organized as specialized lipid rafts [6,7].

Basically, caveolae is formed by the homo- and hetero-

oligomerization of the three currently known caveolins. Of

these caveolins, cav-1 is involved in signal transduction

largely because of the presence of a 20 amino acid micro-

domain which can bind a variety of signal proteins [6], most

often leading to downstream signaling events [8,9]. cav-1 has

been identified as a marker of aggressive prostate carcinoma

[10,11] that promotes progression to the metastatic phenotype

[12]. The significance of cav-1 in cell signaling is demo-

nstrated by its coordinating the interaction, redistribution and

co-localization of several proteins with each other during

experimental clustering of raft components [13]. Typically,

cav-1 co-localizes with androgen receptors within lipid raft

domains to mediate androgen-dependent signals [14,15],

especially downstream signals that modulate the expression

of genes implicated in unregulated cell growth [6].

Unregulated cell growth occurs mostly when cells loose

their ability to undergo apoptosis, often leading to cancer.

Various studies have confirmed the loss in cell growth

control following alteration of apoptotic pathways [16]. The

susceptibility of a cell to apoptosis is widely believed to be

mediated by the expression of a complex family of genes

that principally include p53 [16], which also mediate the

transcriptional activation of cav-1 [17]. Thus, cholesterol-

enrichment which results in oxidative stress [18], often lead

to DNA damage and increase in p53 protein expression [19],

G1 cell cycle arrest and interaction of numerous pro- and

anti-apoptosis proteins [16]. Cholesterol-mediated dysregu-

lation of cav-1 [10], and apoptotic suppressor genes [16] by

aberrant p53 highlights the significance of p53 in

cholesterol-mediated cell growth, and anti-apoptosis. In

all, it is likely that understanding the mechanism of

cholesterol-mediated transcription of apoptosis suppressors

may be significant in elucidating the contribution of sterols

in the etiology, progression or prevention/regression of

prostate cancer.

Overall, the importance of sterols in the promotion of cell

growth may therefore, depend on their orchestrating the co-

localization of cav-1 and androgen receptor at the plasma

membrane [14,15]. Androgen receptors elaborate the

biological effects of androgens in target cells, by mediating

the transcriptional regulation of androgen-regulated genes

[20]. Recent data suggest ethnic variation in the pattern of

expression of a prostate specific androgen-regulated gene

(PCGEM1) [21], which promote unregulated cell growth.

Although this proto-oncogene is irregularly expressed in high

risk and poorly progressed prostate cancer [21], its trans-

criptional regulation by sterols and cav-1 to our knowledge

has not been elucidated. Based on evidence that cav-1 is

differentially transcribed by either sterol status [13,22]

resulting in modulation of cell growth, we hypothesized that

these sterols regulate prostate cell growth by promoting or

repressing the transcription of cav-1 and its downstream

signals. To test this hypothesis, we examined the effects of

different sterol-enrichment on cav-1 expression, cell growth,

apoptosis, and on the expression of downstream proto-

oncogene PCGEM1, using PC-3 and DU145 prostate cancer

cell lines.

2. Materials and methods

2.1. Cell culture

Androgen-independent prostate cancer cell lines PC-3

and DU145 were obtained from ATCC (Manassas, VA). The

cells were cultured in MEM with 10% FBS, 1% penicillin/

streptomycin, 1% glutamine, 1% non-essential amino acids,

0.1% gentamicine and fungizone and buffered with 0.75%

HEPES at 37 8C in 5% CO2 for 24 h. The medium was then

changed to 1% FBS-MEM, and the cells incubated for 24 h

before treatment with sterols. Experimental media were

supplemented with sterols (cholesterol or phytosterols; 10%

campesterol: 75% b-sitosterol) (Acros organics, NJ) at final

concentrations of 16 mM. The phytosterol combination

chosen represent the typical percentage concentration of b-

sitosterol (78–83%) relative to phytosterols found in

peanuts; which is a reported classic phytosterol source

[23]. Optimum sterol concentration of 16 mM was chosen

after dose–response experiments produced results that were

consistent with physiological levels for phytosterols, and

with previously observed values [3,24]. For all treatments,

sterols were mixed in the media with a sterol carrier (2-

hydroxypropyl)-b-cyclodextrin (b-CD) (Sigma–Aldrich, St.

Louis, MO) and supplied to the cells as complexes. To

provide the sterols in an assimilable and nontoxic form, the

sterol to cyclodextrin molar ratio was maintained at 1:300

(16 mM sterol: 5 mM b-CD) as previously reported [24,25].

All treatments were performed in triplicates. After stimula-

tion for 48 h, the cells or total RNA were analyzed in

different experiments.

2.2. Measurement of sterol effects on cell viability

PC-3 and DU145 cells (5000 cells/cm2) were seeded in

triplicates into 6-well plates for 24 h. The medium was

replaced with 1% FBS-MEM for 24 h, then the cells were

supplemented with 16 mM sterols and vehicle for 48 h and

72 h. Viability of cells given different sterol treatment was

measured by trypan blue dye exclusion method. After 48 h,

1 � 106 ml�1 cell suspension for viability assay was

prepared by trypsinization, centrifugation and counting

with a hemocytometer. A 1:1 dilution of 200 ml of the cell

suspension was made using 0.4% trypan blue solution and

G.O. Ifere et al. / Cancer Detection and Prevention 32 (2009) 319–328 321

incubated for 3 min at room temperature. Replicate samples

of stained and unstained cells from each well were counted

with a hemocytometer on a microscope under a 10�objective. Following analysis of variance, data from all

experiments were pooled for further statistical analysis. The

calculated percentage of unstained cells represents the

percentage of viable cells. Micrographs of cells receiving

each of the treatments were taken.

2.3. Cell cycle analysis by flow cytometry

Cell cycle analysis was performed by propidium iodide

(PI) staining. Triplicates of sterol- and vehicle-treated

prostate tumor cells (1 � 10�6) seeded in 6-well cell

cultures dishes were harvested after 48 h and washed with

ice-cold PBS. The cells were then fixed in ice-cold 70%

ethanol, vortexed and stored at 4 8C for 1 h. This was

followed by centrifugation at 3000 rpm for 5 min and two

times washing in PBS. Fifty microliters of RNAse (100 mg/

ml) was added to the pelleted cells and incubated at room

temperature for 15 min. After addition of 400 ml of PI

(50 mg/ml) to the cells, staining was analyzed by flow

cytometry.

2.4. Cell proliferation assay

Cell proliferation was determined using the CellTiter 96

Non-Radioactive Cell proliferation Assay (Promega Corp.,

Madison, WI), which is based on the cellular conversion of a

tetrazolium salt into formazan product that is detectable

using a 96-well plate reader. Briefly, 100 ml of 1 � 104

prostate cells were seeded into wells of a 96-well plate and

incubated for 24 h. Then the medium was changed to one

containing 1% FBS for another 24 h and then supplemented

with vehicle (b-CD), cholesterol or phytosterols (10%

campesterol: 75% b-sitosterol) for 48 h at 37 8C in a

humidified atmosphere. After 72 h of culture, a chromogenic

dye solution (15 ml) was added to each well and the plate

incubated again at 37 8C for 4 h. After incubation, 100 ml of

solubilization solution/Stop Mix was added to each well and

within 1 h, the contents of the wells were mixed to get a

uniformly colored solution. The absorbance of the colored

reaction product was recorded at 570 nm wavelength using

Synergy 2 multi-detection microplate reader (BioTek

Instruments Inc., Winooski, VT).

2.5. RNA isolation and RT-PCR

To determine the effects of sterol treatment on the

expression of cav-1 and PCGEM1 total RNA was extracted

from cultured prostate cancer cells using RNA isolation

protocol according to manufacturer’s instructions (RNeasy

kit, Qiagen, Valencia, CA). To synthesize cDNA by reverse

transcription, 0.25 mg of total RNA and 1 ml each of oligo-dT

and dNTP mix were used as described in the enhanced avian

reverse transcriptase-PCR kit protocol (Sigma–Aldrich, St.

Louis, MO). A portion (2 ml) of each cDNA was used to

amplify the fragments of the genes in the presence of Taq

DNA polymerase (Sigma, MO). Specific primers for RT-PCR

amplification of the various genes were as follows: (a)

PCGEM1: sense 50-AAGTGAGCAGG-CTTGGTGCATTT-

G-30 and anti-sense 50-ACGTGCCTACCCTTAGGAAAG-

CAT-30; (b) cav-1: sense 50-ACCTCAACGATGACGTGGT-

CAAGA-30 and anti-sense 50-TGGAATAGACAC-ACGG-

CTGATGCACT-30; (c) GAPDH: sense 50-CCACCCATGG-

CAAATTCCATGGCA-30 and anti-sense 50-TC-TAGAGG-

GCAGGTCAGGTCCACC-30.Reaction conditions were: 1 cycle of 95 8C for 10 min,

followed by 40 cycles for PCGEM1, cav-1, and GAPDH.

This was followed by heating at 95 8C for 1 and 2 min,

respectively, for denaturation of cav-1, PCGEM1 and

GAPDH. Annealing was at 65 8C and extension at 72 8Cfor 2 min and final extension of 1 cycle of 72 8C for 5 min.

PCR products were analyzed on 1.5% agarose gel and

visualized by ethidium bromide staining method. All

reactions were normalized by GAPDH mRNA expression.

2.6. Assessment of cell apoptosis

The apoptotic and anti-apoptotic effects of phytosterols-,

cholesterol- and vehicle-treated prostate tumor cells seeded

in biological growth chambers (Lab-Tek Instruments, VT)

were assessed according to, the TDT-FragEL DNA

fragmentation method described in kit protocols (Calbio-

chem, San Diego, CA). After staining and counterstaining

with diaminobenzidine (DAB) and methyl green, respec-

tively, an accurate estimate of the overall apoptotic rate was

obtained by acquiring and processing digital images of

stained sections with microimage program (Axio Vision

digital image processing software, Carl Zeiss Microimaging,

Inc., Thornwood, NY). The number of cells staining dark-

brown (apoptosis), and blue-green (total number) were

quantitated independently in five separate fields on three

different slides for each experimental group, and the mean

percentage of apoptotic cells then calculated. Alternatively,

cells treated with vehicle control and different sterols were

washed with ice-cold PBS. To determine the level of

Annexin binding in sterol treated and untreated PC-3 and

DU145 cells, triplicates of all cell treatments were stained

with Alexa Flour 488 annexin V and propidium iodide

following procedures suggested by manufacturer, using the

Vybrant apoptosis assay kit #2 (Molecular Probes Inc.,

Eugene, OR), and then analyzed by flow cytometry using

FACScanTM flow cytometer and CellQuest software (BD

Pharmingen).

2.7. Statistical analysis

Data are given as mean values (�S.D.). Differences

between treatment groups were determined by two-way

ANOVA [26]. In all analyses, a P-value less than 0.05 was

considered statistically significant.

G.O. Ifere et al. / Cancer Detection and Prevention 32 (2009) 319–328322

Fig. 1. Effects of sterol supplementation on viability of PC-3 and DU145

cells. Viability of cells was measured by trypan blue dye exclusion method

after 72 h and the number of cells counted with a hemocytometer on a

microscope under 10� objective. The treatments were assayed in triplicate

examinations.

3. Results

3.1. Cell viability assay

Viability assays were used to estimate the effects of

vehicle, cholesterol and phytosterol treatments on cell

survival. The results obtained did not show any significant

difference (P > 0.05) in the number of viable cells observed

for all the treatments after 48 h (Fig. 1).

3.2. Cell cycle analysis by microscopy

After 48 h of each sterol treatment, the number of cells

undergoing mitosis in a minimum of 20 random microscope

fields was determined from 1 � 105 ml�1 cell suspension.

Representative photomicrographs of cells undergoing each

treatment taken at 48 h show that cholesterol treatment

enhanced mitosis in PC-3 while less mitotic cell population

was observed in phytosterol-treated cells (54 and 29%,

respectively). A similar trend was observed in DU145 cells

(61 and 35%, respectively) (Fig. 2).

3.3. Cell cycle analysis by fluorescent activated cell

sorting

The cell populations were categorized generally as G0/

G1 phase, or quiescent cells (M1), as S phase or cells in the

process of replicating their DNA (M2), and as G2/M phase,

being cells with two full complements of DNA or at mitotic

phase (M3) (Fig. 3). On this basis, we observed a substantial

enrichment of submitotic cell populations in cholesterol-

treated PC-3 and DU145 cells (40.58 and 34.08%,

respectively) than in vehicle treatment (31.66 and

24.57%, respectively). In contrast, the least mitotic cell

subpopulations were observed in both cell lines following

phytosterol treatment (27.71 and 17.37%, respectively). The

highest subpopulations of cells during phytosterol-enrich-

ment of PC-3 and DU145 cells were in the interphase (S

phase or M2) (52.26 and 55.41%, respectively).

Fig. 2. Photomicrographs of cells a

3.4. Effects of sterols on cell proliferation

To assess the effects of different sterols on prostate cell

proliferation and hence neoplasm, we measured the

metabolic activity of viable cells using tetrazolium salt

MTT. Cell proliferation was quantitated by measuring the

absorbance of MTT, as it is reduced to a colored formazan

only by metabolically active and growing cells. Fig. 4A

shows the effect of 72 h sterol supplementation on the

proliferation of PC-3 cells. Cholesterol treatment signifi-

cantly promoted (P < 0.05) cell proliferation as measured

by the absorbance of MTT at 570 nm. Phytosterol treatment

arrested PC-3 cell growth as seen by the proportional growth

of vehicle and phytosterol-treated cells, while a significant

(P < 0.05) increase in cell growth was observed in

cholesterol-treated cells. Similar effects were observed in

DU145 cells (Fig. 4B).

fter 48 h of sterol treatment.

G.O. Ifere et al. / Cancer Detection and Prevention 32 (2009) 319–328 323

Fig. 3. Histograms from flow cytometry demonstrating cycling cell population stained with propidium iodide. PC-3 (A–C) and DU145 cells (D–F) were,

respectively, enriched with vehicle, cholesterol and phytosterol and sorted into different stages of mitosis by flow cytometry. Gate Ml are cells in G0/G1 phase;

gate M2 cells are in S-phase and gate M3 represent cells in G2-M phase.

3.5. Sterol-induced changes in expression of growth

signaling genes

To evaluate the sterol treatment options on the

transcription of up-stream signaling genes and on down-

stream androgen-dependent, proto-oncogenes in cultured

PC-3 and DU145 cell lines, we amplified the genes for cav-

1, and PCGEM1. Control gene expression was performed

with specific primers for GAPDH. The respective amplifica-

tion fragments obtained for cav-1, PCGEM1 and GAPDH

were 276, 537 and 598 bp as predicted (Fig. 5). The intensity

of the mRNA for GAPDH was the same in all the specimens.

The intensity of PCGEM1 bands was higher in cholesterol

than phytosterol-treated cells. The disparity in the pattern of

Fig. 4. Effect of sterol supplementation on proliferation of (A) PC-3 and (B) DU14

and CAMPSIT (Campesterol/p-Sitosterol). Cell proliferation was determined by

formation that is detectable in a 96-well plate reader.

PCGEM1 expression was more pronounced in DU145 cells

than in PC-3 cells (Fig. 5; PCGEM1 lanes 2 and 3 versus

lanes 5 and 6). Remarkably, there was no detectable band in

vehicle (b-CD)-treated samples of both cell lines (Fig. 5,

PCGEM1 lanes 1 and 4). The intensity of cav-1 bands was

higher in phytosterol-treated cells (Fig. 5; cav-1 lanes 3 and

6) than in cholesterol treatment. Differences in the

transcriptional regulation of cav-1 by cholesterol and

phytosterols were more distinct in PC-3 cell lines (Fig. 5;

cav-1 lanes 1–3). cav-1 band intensities were weaker in

vehicle-treated cells.

As shown in Table 1, the relative intensity of PCGEM1

and cav-1 mRNA expressed after scanning densitometry and

normalization to GAPDH indicated that the increase in

5 cells. Cells were treated with: VEH, Vehicle (p-CD); CHOL (Cholesterol)

CellTiter non-radioactive cell proliferation assay based on formazan dye

G.O. Ifere et al. / Cancer Detection and Prevention 32 (2009) 319–328324

Fig. 5. PCR showing the effect of sterols on the expression of PCGEM1 and cav-1. Cyclodextrin (P-CD)-complexed cholesterol or phytosterol (10%

campesterol:75% (3-sitosterol) was added to prostate cancer cells in culture for 2 days. cDNA from cells was amplified by PCR using appropriate primers. RT-

PCR showing expression of genes after sterol treatment on PC-3 (lanes l–3) and on DU145 cells (lanes 4–6). Vehicle treatment (P-CD) lanes 1 and 4; cholesterol

treatment, lanes 2 and 5; phytosterol treatment, lanes 3 and 6.

PCGEM1 expression in cholesterol-enriched PC-3 cells was

no significantly different from the value for phytosterol-rich

PC-3 cells (PCGEM1, 62.56 � 6.71 in cholesterol-rich cells

versus 46.52 � 4.12 in phytosterol-rich cells, P > 0.05).

However, in DU 145 cells, the increase in PCGEM1 gene

expression was significant during cholesterol treatment as

opposed to its level of expression during phytosterol

treatment (PCGEM1, 124.82 � 9.35 in cholesterol-treated

cells versus 44.23 � 7.34 during phytosterol treatment,

P < 0.001). In contrast, the respective increase in cav-1 gene

expression in phytosterol-rich PC-3 or DU145 cells was

significantly higher than observed during cholesterol

supplementation to the same cells (cav-1, 55.25 � 5.19 in

phytosterol-treated PC-3 cells versus 11.32 � 1.55 in

cholesterol-treated cells, P < 0.001 or 34. 54 � 6.23 in

phytsoterol-treated DU145 cells versus 23.98 � 4.76 in

cholesterol-treated cells, P < 0.01).

Table 1

Relative intensity of PCGEM1 and cav-1 mrNA expression after scanning

densitometry and normalization to GAPDH levels.

Gene Cell line Treatment

Vehicle Cholesterol Campsit

Band density of expressed gene (% change)

PCGM1 PC-3 12.12 � 3.15 62.56 � 6.71 46.52 � 4.12

DU145 3.42 � 1.91 124.82 � 9.35 44.23 � 7.34

cav-1 PC-3 0.98 � 0.75 11.32 � 1.55 55.25 � 5.19

DU145 5.42 � 2.36 23.98 � 4.76 34.54 � 6.32

Values are means � standard errors of the means (n = 3) and P < 0.05. n is

number treatments.

3.6. Effects of sterols on cell apoptosis

Unregulated cell growth occurs mostly in cells that have

lost the ability to undergo apoptosis. To validate the role of

apoptosis as a channel for sterol regulation of prostate cell

growth, DNA oligonucloesomes cleaved into detectable

DNA ladder fragments were end-labeled by immunocyto-

chemical procedures using DNA fragmentation detection

kits (Calbiochem, San Diego, CA). Positive control samples

were generated by a procedural step that includes covering

untreated slide-fixed cells with DNAse 1, which fragments

DNA in the cells. Negative controls of the samples were also

generated by substituting deionized water for terminal

deoxynucleotidyl transferase (TDT), thereby preventing

non-specific conjugate binding. After staining and counter-

staining with DAB and methyl green, apoptotic cells showed

dark-brown stained nuclei while non-apoptotic cells showed

blue-green stained nuclei. Representative photomicrographs

showed that in both cell lines, phytosterol treatment was

characterized by greater number of dark-brown staining

nuclei (Fig. 6B and G). There was similarity in the pattern of

blue-green staining observed in photomicrographs of

negative control, vehicle and cholesterol-treated cells of

both cell lines (Fig. 6C–E and H–J). On the other hand, the

dark-brown pattern of staining observed in phytosterol-

treated cells compared with the positive control treatment

(Fig. 6 B and G versus Fig. 6A and F). Calculation of the

mean percentage of apoptotic cells using the TDT-FragEL

DNA fragmentation experiment shows significant increase

in apoptosis within positive control and phytosterol-treated

G.O. Ifere et al. / Cancer Detection and Prevention 32 (2009) 319–328 325

Fig. 6. Immunocytochemical analysis of PC-3 (A–E) and DU145 cells (F–J) stained with TdT FragEL DNA Fragmentation detection kit. Apoptotic cell nuclei

(positive control and phytosterol) stained dark-brown while non-apoptotic cell nuclei (negative control, vehicle and cholesterol) stained blue-green. (For

interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Table 2

Comparison of percentage of annexin positive cells after treatment with

sterols.

Cell line Treatment

Vehicle Cholesterol Campsit

% annexin V-positive cells

PC-3 0.97 � 0.43 0.86 � 0.72 2.93 � 0.45

DU145 4.74 � 1.22 4.71 � 1.74 7.64 � 1.87

Results are expressed as means � standard deviations (n = 3), where n is the

number of treatments. Differences between groups were considered sig-

nificant when P < 0.05.

cells as compared to negative control, vehicle- and

cholesterol-treated cells (P < 0.001) (Fig. 7). To confirm

that phytosterol-mediated cell death is an apoptotic process,

sterol-treated and control cells were assayed for the

translocation of phosphatidylserine from the inner to the

outer leaflet of the plasma membrane using annexin V

affinity assay. Test samples with higher levels of annexin V

binding were considered positive and apoptotic. Dead cells

were stained by PI, which enters cells with disrupted plasma

membrane and binds to DNA. In Fig. 8, the cell populations

were represented by R1 for dead cells, R2 for live cells and

R3 for apoptotic cells. Following sterol treatment, FACS

analysis of typical groups showed that phytosterol treatment

(data Fig. 8C and F) induced higher percentage of apoptotic

cells (2.82 and 6.37%) when compared to other treatment

options. The mean percentage of apoptotic cells was

2.93 � 0.45 and 7.64 � 1.87% for phytosterol-treated PC-

3 and DU145 cells, respectively (Table 2).

Fig. 7. Effects of sterols on apoptosis IN PC-3 and DU145 cells. Graph

represents the means � S.D. for three independent experiments shown in

Fig. 6A. Positive C Campsit and cholesterol groups have a P < 0.05 in

comparison with the value for negative control cells. Positive C and negative

C represent positive and negative control groups, respectively.

4. Discussion

This study describes the function of sterols in the

expression of downstream regulatory genes for prostate cell

growth. Also cell viability during various sterol treatment

options was assayed in order to ascertain their toxicity. The

calculated percentage viability show neither adverse cell

viability nor significant necrosis arose from all the treatment

options. This treatment regimen was chosen because

previous studies showed that, direct exposition of myco-

plasma cells to comparable amount of vehicle (b-CD)

presently used, enabled unimpeded cell growth [25]. That

investigation also concluded that b-CD favorably combines

nontoxicity with a proper binding capacity and so, suggested

as a safe and efficient injectable delivery system for poorly

soluble drugs [25]. Interestingly, micrographs of cells

undergoing our sterol treatment options exhibit different

stages of the cell cycle. These photomicrographs and flow

cytometric histograms revealed more mitotic appearance for

cholesterol-enriched cells than for phytosterol-treated cells.

The reduced mitotic cell subpopulation observed in

phytosterol-enriched cells and the corresponding large

number of within the interphase suggests the induction of

these cells for G1/S phase arrest of the cell cycle in readiness

for apoptosis. This corroborates reports that, induction of

G.O. Ifere et al. / Cancer Detection and Prevention 32 (2009) 319–328326

Fig. 8. Determination of apoptosis by flow cytometry. After treatment of PC-3 (A–C) and DU145 (D–F) cells with sterols, the extent of apoptosis was assessed

by annexin V/propidium iodide assay. The highest percentage of apoptosis is found in phytosterol-treated PC-3 and DU145 cells (trisector R3 of C and F). There

was, respectively, no difference in percentage of apoptosis in vehicle-treated cells (A and D), and cholesterol-treated cells (B and E).

apoptosis is associated with the repression of genes that

influence cell cycle progression or genes that directly

regulate cell survival [27]. Furthermore, our observation of

mitosis in most cholesterol-treated cells is consistent with

previous demonstration of cholesterol requirement in cell

cycle progression from G2 to M phase [28]. Our findings that

phytosterols effectively inhibit growth of PC-3 and DU145

cells by inducing G1/S phase arrest is supported by previous

studies on various tumors of different origin [24]. One of the

reasons given for phytosterol/b-sitosterol-mediated inhibi-

tion of cell growth is its activation of MAPK (ERK1/2) [24].

Although MAPK pathway is central in the integration of

many extracellular and intracellular signals [24], little is

known about its link with sterol-modulated genes that have a

direct growth promoting function, and their manner of

transducing cell growth or apoptosis.

MTT assay showed that both cell lines used in these

studies were stimulated to cell proliferation by cholesterol

treatment. Previous studies further showed that cholesterol

promotes tumor invasiveness and cell binding to collagen IV,

a protein implicated in invasive process [29]. MTT assays

specifically measure states of metabolic activity, as

proliferating cells are metabolically more active than non-

proliferating or resting cells [30]. The observed promotion

of cell proliferation by cholesterol as measured by increased

metabolic activity, supports known cholesterol functions,

which include the regulation of membrane-bound proteins,

enzymes and several signal transduction pathways [24].

MTT assay in this study showed no difference in cell growth

for phytosterol- and vehicle-treated cells. Even so, the

significant reduction in proliferation of phytosterol-treated

cells as compared to cholesterol treatment reveals the

inhibitory effect of phytosterols on prostate cell growth.

Again, this study uniquely demonstrated the modulation of

growth-regulating genes by sterols. Cholesterol-enrichment

triggered overexpression of PCGEM1 in our cultures. The

differential expression of this gene by the different sterol

treatment options was more distinct in DU145 than in PC-3

cells. This indicates differential regulatory patterns of this

gene by sterols across the cell lines. Our inability to detect

PCGEM1 bands in vehicle treatment across both androgen-

independent cell lines was remarkably demonstrated by

Srinkantan et al. [31]. They showed that PCGEM1 expression

was detectable only in androgen receptor-positive cell lines

such as LNCap. Thus, our study uniquely demonstrates that

cholesterol upregulates PCGEM1 expression. Currently, there

is no known explanation for this observed cholesterol ‘‘turn

on’’ of this seemingly shut androgen-dependent gene in

androgen receptor-negative prostate cell lines. Nevertheless,

we observed that the ‘‘turn on’’ of this proto-oncogene

corresponds with cholesterol-dependent attenuation of cav-1

gene expression versus its upregulation by phytosterols. The

attenuation of cav-1 expression and the corresponding

overexpression of known androgen-dependent gene reinforce

previous observation that, reduced expression of cav-1 by

anti-sense cDNA converts androgen-insensitive metastatic

prostate cancer cells into androgen-sensitive cells [32]. This

though, is consistent with recent observation that cav-1

expression is greatly reduced in most (but not all) onco-

genically transformed and human cancer cells [33]. This may

be based on existing paradigm those cav-1 functions as a

tumor suppressor protein that inhibits anchorage-independent

G.O. Ifere et al. / Cancer Detection and Prevention 32 (2009) 319–328 327

growth and matrix invasiveness in human cancer cells [33].

Despite abundant evidence that cav-1 has tumor transforma-

tion properties, recent study conflicts with these findings,

suggesting it has tumor promoter function [34]. This was

based on demonstration that downregulation of cav-1

expression by small interfering RNA approach, significantly

reduced the tumorigenic and metastatic potential of mouse

model prostate cancer cells [34]. This is supported by further

evidence showing that overexpression of cav-1 was associated

with metastatic progression as well as androgen insensitivity

in low caveolin, androgen-sensitive mouse prostate cancer

cells [34,35]. Using androgen-insensitive prostate cancer cell

lines we rather observed that phytsoterol-induced high

expression of cav-1 corresponds with a reduced expression

of the protoncogene, PCGEM1 in contrast to the cholesterol-

induced pattern of expression of these genes. This rather

agrees with the model suggesting that cav-1 is a growth

suppressor, and may enhance comparatively lower androgen

sensitivity during phytosterol treatment as opposed to cho-

lesterol treatment How attenuated cav-1 expression elaborates

androgen-dependent signaling is yet to be fully understood.

However, sufficient evidence suggests that this contradiction

in cav-1 activity derive from its function as a scaffolding

protein that interacts with, and regulates a variety of signaling

pathways that include proto-oncogenes [9] like PCGEM1.

Specifically, cholesterol reportedly induces the interaction

of cav-1 with androgen receptor, thus triggering the trans-

cription of some downstream genes [15]. A classic case is the

transient co-localization of cav-1 with androgen receptors

in lipid-enriched rafts, and the facilitation of androgen-

dependent signaling [14,15]. We thus identify a putative

mechanism for cholesterol induction of overexpressed

PCGEM1, an androgen-dependent proto-oncogene.

Translationally, high expression of PCGEM1 has been

reported to correlate with cell growth in human prostate

cancer, suggesting its potential as biomarker for high-risk

prostate cancer [21]. Environmental factors among them

high cholesterol diets [36] are reported risk factors for

prostate cancer in African-Americans [37]. Thus over-

expression of PCGEM1 following cellular cholesterol-

enrichment further supports its biomarkers status for high-

risk prostate cancer.

Our unique demonstration of downregulated PCGEM1

expression by phytosterols is comparable to various reports

showing phytosterol suppression of cell growth [24].

Overall, our result suggests that phytosterols play a role

in the prevention or reversal of prostate neoplasm by

suppressing PCGEM1 mRNA expression. The mechanism

for reversal of prostate cell growth by underexpressed

PCGEM1 may be deduced indirectly from very recent

findings that, overexpression of PCGEM1 in LNCap cell

culture model inhibits experimental apoptosis [38]. This is

supported by our flow cytometric analysis showing that

phytosterols-induced apoptosis, besides underexpression of

PCGEM1. Overexpression of this oncogene and inhibition

of apoptotic machinery during cholesterol enhancement

agrees with the current paradigm in cancer cell biology that,

defects in normal apoptotic mechanisms play a major role in

the pathogenesis of various cancers [39].

In all, there are various conceptual frameworks for the

mechanism of inhibition of cellular apoptosis by cholesterol.

One of them is based on the relationship between cholesterol-

enrichment, cav-1 expression and mediation of downstream

signaling events. Overexpression of cav-1 has been found to

regulate the activity of PI3K [15], which trigger downstream

protein kinase Akt-dependent anti-apoptosis [40]. Cholesterol

is essential in the arrangement of cav-1 within rafts [6,7], and

may provide support for caveolin1-PI3K-Akt activation

cascade of anti-apototic signals. Besides, our study shows

the attenuation of cav-1 gene expression by cholesterol

loading, thus revealing a new conceptual framework for

cholesterol promotion of cell growth or anti-apoptosis based

on the ability of attenuated cav-1 to elaborate androgen

sensitivity.

Further support for sterol regulation of growth or apoptosis

in prostate cancer cells was examined by immunocytochem-

ical analysis of PC-3 and DU145 cells. Photomicrographs of

end-labeled DNA ladder fragments indicated that, phytos-

terol-treated cells were characterized by greater number

of detectable DNA ladder fragments, which characterize

apoptosis. Less apoptosis signals were observed in cholesterol

and vehicle-treated cells. Our data on immunocytochemistry,

flow cytometry and transcriptional analysis of apoptotic and

anti-apoptotic genes confirm that phytosterols inhibit growth

of prostate cell lines by induction of apoptosis.

In summary, our study revealed that sterols initiate

differential expression of PCGEM1 and cav-1 in prostate

cancer cell lines. We also showed that cholesterol ‘‘turned

on’’ seemingly shut androgen-dependent downstream proto-

oncogene (PCGEM1) in androgen receptor-negative pros-

tate cell lines. The integration of sterol responsive PCGEM1

and cav-1 expression, with cell growth or apoptosis reveals a

novel pathway for sterol regulation of prostate neoplasm or

apoptosis.

We conclude that differential activation of various

neoplastic or apoptotic genes by different sterol treatment

options may enable the elucidation of prostate neoplasm at

the molecular level so as to develop effective prevention and

intervention strategies for control and prevention of the

malignancy. In view of the heterogeneous nature of human

prostate cancer, the sterol responsive gene PCGEM1 may

efficiently predict neoplasms that would respond to

chemoprevention by phytosterols. Also, elucidating the

mechanism by which cholesterol mediate prostate cancer

would strengthen the existing dogma that dietary cholesterol

plays a role in the disparity in prostate cancer incidence

along ethnic lines.

Conflict of interest

None.

G.O. Ifere et al. / Cancer Detection and Prevention 32 (2009) 319–328328

Acknowledgements

This research was supported by NIH Grants#:

5P20MD002285-02, GM08247, A141231. The authors

are grateful to Clark Atlanta University RCMI Program

and the Molecular Biology Core facility laboratory.

References

[1] Platz EA, Rimm EB, Willett WC, Kantoff PW. Racial variation in

prostate cancer incidence and in hormonal system markers among

male health professionals. J Natl Cancer Inst 2000; 92:2009–20017.7.

[2] Hayes RB. Are dietary fat vasectomy risk factors for prostate cancer? J

Natl Cancer Inst 1995; 87:629–31.

[3] Awad AB, Fink CS. Phytosterols as anticancer dietary components:

evidence and mechanism of action. J Nutr 2000; 130:2127–30.

[4] Von Holtz RL, Fink CS, Awad AB. b-Sitosterol activates the sphin-

gomyelin cycle and induces apoptosis in LNCap human prostate

cancer cells. Nutr Cancer 1998; 32:8–12.

[5] Arora A, Raghuraman H, Chattopadhay A. Influence of cholesterol

and ergosterol on membrane dynamics: a fluorescence approach.

Biochem Biophys Res Commun 2004; 318:920–6.

[6] Daniel EE, El-Yazbi A, Cho WJ. Caveolae and calcium handling, a

review and a hypothesis. J Cell Mol Med 2006; 10:529–44.

[7] Zhuang L, Kim J, Adam RM, Solomon KR, Freeman MR. Cholesterol

targeting alters lipid raft composition and cell survival in prostate

cancer cells and xenografts. J Clin Invest 2005; 115:959–68.

[8] Williams TM, Lisanti MP. The caveolin genes: from cell biology to

medicine. Ann Med 2004; 36:584–95.

[9] Cohen AW, Hnasko R, Schubert W, Lisanti MP. Role of caveolae and

caveolins in health and disease. Physiol Rev 2004; 84:1341–79.

[10] Yang G, Truong LD, Wheeler TM, Thompson TC. Caveolin-1 expres-

sion in clinically confined human prostate cancer: a novel prognostic

marker. Cancer Res 1999; 59:5719–23.

[11] Yang G, Addai J, Ittmann M, Wheeler TM, Thompson TC. Elevated

caveolin-1 levels in African-American versus white-American pros-

tate cancer. Clin Cancer Res 2000; 6:3430–3.

[12] Li L, Yang G, Ebara S, Satoh T, Nasu Y, Timmen TL, et al. Caveolin-1

mediates testosterone-stimulated survival/clonal growth and promotes

metastatic activities in prostate cancer cells. Cancer Res 2001;

61:4386–92.

[13] Brown DA, London E. Structure and function of sphingolipid- and

cholesterol-rich membrane rafts. J Biol Chem 2000; 275:17221–4.

[14] Razandi M, Alton G, Pedram A, Ghonshani S, Webb P, Levin ER.

Identification of a structural determinant necessary for the localization

and function of estrogen receptor alpha at the plasma membrane. Mol

Cell Biol 2003; 23:1633–46.

[15] Lu ML, Schneider MC, Zheng Y, Zhang X, Richie JP. Caveolin-1

interacts with androgen receptor, a positive modulator of androgen

receptor mediated transactivation. J Biol Chem 2001; 276:13442–51.

[16] Bruggers CS, Fults D, Perkins SL, Coffin CM, Carroll WL. Coex-

pression of genes involved in apoptosis in central nervous system

neoplasms. Pediatr Hematol Oncol 1999; 21:19–25.

[17] Bist A, Fielding CJ, Fielding PE. P53 regulates caveolin gene tran-

scription, cell cholesterol and growth by a novel mechanism. Bio-

chemistry 2000; 39:1966–72.

[18] Homma Y, Kondo Y, Kaneko M, Kitamura T, Nyou WT, Yanagisawa

M, et al. Promotion of carcinogenesis and oxidative stress by dietary

cholesterol in rat prostate. Carcinogenesis 2004; 25:1011–4.

[19] Chen K, Albano A, Ho A, Keaney Jr JF. Activation of p53 by oxidative

stress involves platelet-derived growth factor-b receptor-mediated

ataxia telangiectasia mutated (ATM) kinase activation. J Biol Chem

2003; 278:39527–33.

[20] Segawa T, Nau ME, Xu LL, Chilukuri RN, Makarem M, Zhang W,

et al. Androgen-induced expression of endoplasmic reticulum (ER)

stress response genes in prostate cancer cells. Oncogene 2002;

21:8749–58.

[21] Petrovics G, Zhang W, Makarem M, Street JP, Connely R, Sun L, et al.

Elevated expression of PCGEM, a prostate-specific gene with cell

growth-promoting function, is associated with high risk prostate

cancer patients. Oncogene 2004; 23:605–11.

[22] Metzler B, Hu Y, Dietrich H, Xu Q. Increased expression and activa-

tion of stress-activated protein kinase/c-Jun NH2- terminal protein

kinases in atherosclerotic lesions coincide with p53. Am J Pathol 2000;

156:1875–86.

[23] Awad AB, Chan KC, Downie AC, Fink CS. Peanuts as a source of b-

sitosterol, a sterol with anticancer properties. Nutr Cancer 2000;

36:238–41.

[24] Awad AB, Williams H, Fink CS. Effect of phytosterols on cholesterol

metabolism and MAP kinase in MDA-MB-231 human breast cancer

cells. J Nutr Biochem 2003; 14:111–9.

[25] Greenberg-Ofrath N, Terespolosky Y, Kahane I, Bar R. Cyclodextrins

as carriers of cholesterol and fatty acids in cultivation of mycoplasmas.

Appl Environ Microbiol 1993; 59:547–51.

[26] Bishop ON. Statistics for biology . Microcomputer. London: Longman

(EF) Ltd., 1985.

[27] Stromblad S, Becker JC, Yebra M, Brooks PC, Cheresh DA. Sup-

pression of p53 activity and p21-WAF1/CIP1 expression by vascular

cell integrin avb3 during angiogenesis. J Clin Invest 1996; 98:

426–33.

[28] Martinez-Botas J, Suarez Y, Ferruelo AJ, Gomez-Coronado D, Lasun-

cion MA. Cholesterol starvation decreases P34cdc2 kinase activity and

arrest the cell cycle at G2. FASEB J 1999; 13:1359–70.

[29] Awad AB, Fink CS, Williams H, Kim U. In vitro and in vivo (SCID

mice) effects of phytosterols on the growth and dissemination of

human prostate cancer PC-3 cells. Eur J Cancer Prev 2001; 10:507–13.

[30] Corban-Weilhelm H, Ehemann V, Becker G, Greulich D, Braun K,

Debus J. Comparison of different methods to assess the cytotoxic

effects of cytosine deaminase and thymidine kinase gene therapy.

Cancer Gene Ther 2004; 11:208–14.

[31] Srinkantan V, Zhiqiang Z, Petrovics G, Xu L, Augustus M, Davis L,

et al. PCGEM1, a prostate-specific gene, is overexpressed in prostate

cancer. Proc Natl Acad Sci USA 2000; 97:12216–21.

[32] Goh M, Chen F, Paulsen MT, Yeager AM, Dyer ES, Ljungman M.

Phenylbutyrate attenuates the expression of Bcl-XL, DNA-PK, Caveo-

lin-1, and VEGF in prostate cancer cells. Neoplasia 2001; 3:331–8.

[33] Fiucci G, Ravid D, Reich R, Liscovitch M. Caveolin-1 inhibits

anchorage-independent growth, anoikis and invasiveness in MCF-7

human breast cancer cells. Oncogene 2002; 21:2365–75.

[34] Williams TM, Hassan GS, Li J, Cohen AW, Medina F, Frank PG, et al.

Caveolin-1 promotes tumor progression in an autochthonous mouse

model of prostate cancer: genetic ablation of cav-1 delays advanced

prostate tumor development in tramp mice. J Biol Chem 2005;

280:25134–45.

[35] Thompson CT. Metastasis-related genes in prostate cancer: the role of

caveolin-1. Cancer Metast Rev 1999; 17:439–42.

[36] Freeman MR, Solomon KR. Cholesterol and prostate cancer. J Cell

Biochem 2004; 91:54–69.

[37] Reddy S, Shapiro M, Morton R, Brawley OW. Prostate cancer in black

and white Americans. Cancer Metast Rev 2003; 22:83–6.

[38] Fu X, Ravindranath L, Tran N, Petrovics G, Srivastava S. Regulation

of apoptosis by a prostate-specific and prostate cancer-associated

noncoding gene, PCGEM1. DNA Cell Biol 2006; 25:135–41.

[39] Banerjee P, Banerjee S, Brown TR. Bcl-2 protein expression correlates

with cell survival and androgen independence in rat prostatic lobes.

Endocrinology 2002; 143:1825–32.

[40] Rusinol AE, Thewke D, Liu J, Freeman N, Panini SR, Sinensky MS.

AKT/Protein kinase B regulation of BCL family members during

oxysterol-induced apoptosis. J Biol Chem 2004; 279:1392–9.