Combined effects of terazosin and genistein on a metastatic hormone-independent human prostate cancer cell line.

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Combined effects of terazosin and genistein on a metastatic,

hormone-independent human prostate cancer cell line

Kee-Lung Chang

a

, Hsiao-Ling Cheng

b

, Li-Wen Huang

c

, Bau-Shan Hsieh

b

, Yu-Chen Hu

b

,

Tsai-Tung Chih

d

, Huey-Wen Shyu

d

, Shu-Jem Su

d,*

a

Department of Biochemistry, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan b

Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan c

Department of Medical laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung 80708, Taiwan d

Department of Medical Technology, Bachelor Degree Program of Health Beauty, School of Medicine and Health Sciences, FooYin University, No. 151, Chinhsueh Rd., Ta-liao, Kaohsiung 83101, Taiwan

a r t i c l e

i n f o

Article history: Received 26 April 2008

Received in revised form 25 September 2008 Accepted 21 October 2008 Keywords: Genistein Terazosin Prostate cancer Apoptosis

Vascular endothelial growth factor (VEGF)

a b s t r a c t

Metastatic prostate cancer progresses from androgen-dependent to androgen-indepen-dent. Terazosin, a long-acting selective

a

1-adrenoreceptor antagonist, induces apoptosis of prostate cancer cells in an

a

1-adrenoreceptor-independent manner, while genistein, a major soy isoﬂavone, inhibits the growth of several types of cancer cells. The present study was designed to test the therapeutic potential of a combination of terazosin and genistein using a metastatic, hormone-independent prostatic cancer cell line, DU-145.

Terazosin or genistein treatment inhibited the growth of DU-145 cells in a dose-depen-dent manner, whereas had no effect on normal prostate epithelial cells. Addition of 1

l

g/ml of terazosin, which was inactive alone, augmented the growth inhibitory effect of 5

l

g/ml of genistein. Co-treatment with terazosin resulted in the genistein-induced arrest of DU-145 cells in G2/M phase being overridden and an increase in apoptotic cells, as evidenced by procaspase-3 activation and PARP cleavage. The combination also caused a greater decrease in the levels of the apoptosis-regulating protein, Bcl-XL, and of VEGF165 and VEGF121than genistein alone.

In conclusion, the terazosin/genistein combination was more effective in inhibiting cell growth and VEGF expression as well as inducing apoptosis of the metastatic, androgen-independent prostate cancer cell line, DU-145, than either alone. The doses used in this study are in lower and nontoxic anticancer dosage range, suggesting this combination has potential for therapeutic use.

1. Introduction

Prostate cancer is a leading cause of cancer-related

deaths in men

[1,2]

. Mortality results from metastasis to

the bone and lymph nodes and progression from

andro-gen-dependent to androgen-independent prostatic growth

[3]

. Radiation therapy is curative for localized disease, but

there is no treatment for metastatic prostate cancer

[1]

.

Terazosin is a long-acting selective

a

1-adrenoceptor

antag-onist that is used clinically to provide acute relief of the

obstructive symptoms associated with benign prostatic

hypertrophy (BPH)

[4–6]

and recent studies have shown

that it induces apoptosis of prostate epithelial and smooth

muscle cells in patients with BPH

[7–10]

. It also induces

apoptosis of prostate cancer cells via an

a

1-adrenorecep-tor-independent mechanism

[11–17]

and has

anti-angio-genic effects in the human prostate

[18–21]

. These

ﬁndings provide the rationale for the development of an

effective therapeutic strategy using terazosin for patients

* Corresponding author. Tel.: +886 7 781 1151x5412; fax: +886 7 397 2257.

E-mail address:sc096@mail.fy.edu.tw(S.-J. Su).

Contents lists available at

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Cancer Letters

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with androgen-dependent or androgen- independent

pros-tate cancer.

Epidemiological studies have shown that, in Asia, the

decreased occurrence of cancers, including prostate cancer,

is associated with consumption of soy

[22,23]

. Soy

isoﬂav-ones are natural chemoprotectors against cancer and are

not toxic for normal cells

[24]

; genistein (5,7,4

0

-trihydr-oxyisoﬂavone) is one of the predominant compounds of

soy isoﬂavones

[24,25]

. Genistein inhibits cell growth both

in several types of cancer, including prostate

[26,27]

,

breast

[28,29]

, lung

[30]

, bladder

[31,32]

, and liver

[33,34]

cancers, and in BPH

[35]

and inhibits angiogenesis

in tumors

[36]

.

In an attempt to reduce the therapeutic dosage of

tera-zosin so as to decrease its toxicity in prostate cancer

treat-ment, we tested the effect of combining it with genistein,

using a hormone-independent prostate cancer cell line,

DU-145. The anti-angiogenesis and apoptosis-related

pro-teins are considered to be the common downstream

effec-tors mediating the effects of terazosin and isoﬂavones in

prostate cancer, those were examined to evaluate

thera-peutic efﬁcacy.

2. Materials and methods

2.1. Cell culture and viability assay

The DU-145 cell line, an androgen-independent tumor

cell type, derived from a human prostate carcinoma, was

obtained from the American Type Culture Collection

(Rock-ville, MD). The cells were maintained in MEM (GibcoBRL,

Grand Island, NY) containing 10% fetal bovine serum

(Gib-coBRL, Grand Island, NY). Normal human prostate

epithe-lial cell (PrEC) was obtained from Clonetics (San Diego,

CA) and maintained according to the manufacturer’s

instructions using PrEGM medium. PrEC cells were used

at passage 3–6.

Cells were seeded in each well of a 24-well culture plate

(Corning, New York, USA) and grown at 37 °C in a 5% CO

2

incubator. After 24 h incubation, the cells were treated

with terazosin (Sigma, St. Louis, MO) and/or genistein

(Gib-coBRL, Grand Island, NY) for 3 days, and then cell number

was counted with crystal violet elution assay for viability,

and expressed as a percentage of that of the corresponding

control group. Terazosin was dissolved in distilled water.

Genistein was dissolved in dimethyl sulfoxide (DMSO,

Sig-ma, St. Louis, MO), the ﬁnal DMSO concentration being less

than 0.5% (v/v); the same concentration of DMSO was

added to the controls.

2.2. Cell cycle analysis

For 48 h with or without terazosin and/or genistein

treatment, the distribution of cells in different stages of

the cell cycle was estimated by ﬂow cytometric DNA

anal-ysis, as described previously

[31]

. A minimum of 1 10

4

cells per sample was evaluated by a Elite-Esp ﬂow

cytom-etry (Miami FL, US) in each case. The percentage of cells in

each cell cycle phase (Sub-G1, G0/G1, S, or G2/M) was

cal-culated using Cell FIT research software (Becton-Dickinson,

Mountain View, CA).

2.3. Detection of apoptosis by ﬂow cytometry and

ﬂuorescence microscopy

TUNEL staining was performed following the protocol

recommended in the commercial kit (Boehringer,

Mann-heim, Germany). Apoptotic cells were also detected by

ﬂuorescence microscopy using Hoechst 33342 dye (Sigma,

St. Louis, MO) to label the nuclei and propidium iodide to

stain DNA as described previously

[37]

.

2.4. Western blot analysis

Cytosolic extracts were prepared from cells and the

protein in the supernatant was quantiﬁed using a protein

assay kit (Bio-Rad Laboratories, Hercules, CA). A sample

(60

l

g) was electrophoresed on 12% SDS–polyacrylamide

gels then transferred to nitrocellulose membranes. The

rabbit

polyclonal

antibodies

against

human

PARP,

Concentration (

μg/ml )

0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80

Cell number (

% of

control

)

Cell number (

% of

control

)

Cell number (

% of

control

)

0 20 40 60 80 100 120 Terazosin (T) Genistein (G)

A

B

C

DU-145

Concentration (

μg/ml )

0 20 40 60 80 100 120 Terazosin (T) Genistein (G) PrEC C T1 G5 T1+G5 0 20 40 60 80 100 120 DU-145

Fig. 1. Effects of genistein and terazosin on DU-145 and PrEC cell growth. Triplicate samples of cells were incubated for 3 days with different concentrations of genistein (G) or terazosin (T) alone (A and B) or in combination (C).*

P < 0.05 compared to the untreated group.# P < 0.05 compared to the genistein-treated group.

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Bcl-X

S

/

L

, procaspase-3, or b-actin or mouse monoclonal

anti-human Bcl-2 and Bax antibody (Santa Cruz, CA,

USA) was used as the ﬁrst antibody and followed by

the appropriate horseradish peroxidase-labeled

second-ary antibody (PharMingen, San Diego, CA, USA). The

bound antibody was quantiﬁed by chemiluminescence

detection (PerkinElmer Life Sciences, Inc.). b-Actin was

used as the internal control. The amount of the protein

of interest, expressed as arbitrary densitometric units,

was normalized to the densitometric units for b-actin,

then the density of the band was expressed as the

rela-tive density compared to that in untreated cells (control),

taken as 100%.

2.5. Reverse transcriptase polymerase chain reaction

(RT-PCR)

After total cellular RNA was extracted, the

comple-mentary DNA (cDNA) was synthesized. Then, polymerase

chain reaction (PCR) was performed using a human VEGF

genes set-3 multiplex PCR kit (MBI, So. San Francisco,

USA) and cDNA ampliﬁcation was performed according

to the manufacturer’s procedure. The ampliﬁed PCR

products were separated by gel electrophoresis in 2%

agarose, the intensity of each band calculated by

densi-tometry analysis, and the results expressed as a

percent-age of the density of the corresponding b-actin gene

band.

2.6. VEGF secretion assay

The cells (2 10

5

_{) were incubated overnight at 37 °C in}

6 cm plastic dishes, then for another 36 h with or without

terazosin and/or genistein in MEM containing 10% fetal

bo-vine serum. After incubation, the medium was collected

and the concentrations of VEGF were measured using an

ELISA kit (R&D, Wiesbaden, Germany) according to the

manufacturers’ instructions.

2.7. Statistical analysis

All experiments were repeated for three times. All data

are presented as means ± SD. Differences in cell cycle

dis-tribution were analyzed using the x

2

_{test. Other differences}

were analyzed by analysis of variance, and a Scheffe test

was used to identify differences between the individual

means. Statistical analyses were performed using SAS

(ver-sion 6.011; SAS Institute Inc., Cary, NC). A p value of <0.05

was considered statistically signiﬁcant.

3. Results

3.1. Effects on cell growth

To gain an initial insight into the effects of terazosin or genistein, alone or in combination, on human prostate cancer cells and normal epithelial cells, DU-145 and PrEC cells were treated for 3 days without or with different doses of terazosin and/or genistein. For 3 days

incuba-Fig. 2. Genistein and terazosin induce apoptosis of DU-145 cells. (A) Exponentially growing cells were treated for 48 h and evaluated by TUNEL staining. The gray peak is the vehicle control.*

P < 0.05 compared to the untreated group.#

P < 0.05 compared to the genistein-treated group. (B) Cells were stained using Hoechst 33342 and propidium iodide. C: untreated cells, G5: genistein 5

l

g/ml, T1 + G5: terazosin 1

l

g/ml and genistein 5

l

g/ml.

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tion, the untreated DU-145 cell proliferated by 2.3-fold, whereas terazo-sin or genistein alone inhibited cell growth in a dose-dependent man-ner, genistein being much more effective than terazosin (Fig. 1A); 5

l

g/ml of genistein resulted in more than 50% inhibition and was cho-sen for use in the combination tests. But there was no effect of terazo-sin or genistein on PrEC cells (Fig. 1B) under these dosage ranges. Although 1

l

g/ml of terazosin had no signiﬁcant effect on DU-145, the combination of 1

l

g/ml of terazosin and 5

l

g/ml of genistein was more effective in growth inhibition than 5

l

g/ml of genistein alone (Fig. 1C). However, no further increase in cell growth inhibition was seen when the dose of terazosin in the combination was increased up to 20

l

g/ml (data not shown). Moreover, the combination of 1

l

g/ml of terazosin and 5

l

g/ml of genistein had no inhibition of cell growth in the PrEC cells (data not shown). We therefore chose this combination dosage for study.

3.2. Apoptosis induction

Apoptotic cells were assessed by flow cytometric analysis after TUNEL staining (Fig. 2A). After 48 h treatment with of genistein, a significant in-crease in the percentage of TUNEL-positive apoptotic cells was seen com-pared to controls. Although terazosin alone did not cause a significant change in the number of apoptotic cells, a stronger effect was observed with combined treatment, the combination resulting in a significant in-crease in the percentage of apoptotic cells compared to genistein alone (20.13 ± 3.61 vs. 29.54 ± 3.12; p < 0.05). The treatment-induced apoptosis was also apparent from the morphologic changes detected by fluores-cence microscopy of Hoechst 33342 dye and propidium iodide-labeled cells (Fig. 2B).

3.3. Cell cycle arrest

To gain an insight into the effects on cell cycle distribution, DU-145 cells were incubated for 48 h with genistein and/or terazosin. As shown

inFig. 3, terazosin had no signiﬁcant effect on the cell cycle distribution, but genistein caused cell arrest in G2/M phase. Surprisingly, the addition of terazosin to genistein overrode the G2/M phase arrest (percentage of cells in G2/M phase 19.64 ± 2.03% compared to 29.66 ± 2.98%) and in-creased the percentage of cells in sub-G1 phase from 14.26 ± 2.16% to 22.97 ± 2.48%, but did not signiﬁcantly change the percentage of cells in G0/G1 or S phase.

3.4. Procaspase-3 activation and PARP cleavage

Caspase-3, a member of the caspase family, is expressed in cells as an inactive 32 kDa proenzyme, 3. During apoptosis, procaspase-3 is activated by cleavage at speciﬁc Asp residues to generate active cas-pase-3, consisting of 17 and 12 kDa subunits. Caspase-3 then cleaves its substrate, PARP, into 85 and 24 kDa fragments. As shown in the immuno-blots inFig. 4. Thirty-six hours treatment of DU-145 cells with genistein alone or terazosin/genistein resulted in a marked decrease in procaspase-3 (Fig. 4A), the terazosin/genistein combination being more effective. In accordance with the activation of procaspase-3, signiﬁcant generation of the 85 kDa PARP cleavage fragment was seen with all treatments, including terazosin alone (Fig. 4B). These results show that active cas-pase-3 was generated by either terazosin or genistein and that the com-bination was more effective than either alone.

3.5. Expression of apoptosis-related proteins

To determine whether the treatment-induced apoptosis was associ-ated with altered expression of apoptosis-regulating proteins, DU-145 cells were treated for 36 h with genistein and/or terazosin, then were subjected to Western blotting.Fig. 5shows that terazosin had no effect on Bcl-XLlevels, whereas genistein caused a marked decrease, and the combination was even more effective. Effects on Bcl-2 could not be tested, as it was undetectable in DU-145 cells, in accordance with a previous re-port[38]. There was no signiﬁcant difference in the pro-apoptotic protein

Fig. 3. Effect of genistein and/or terazosin on DU-145 cell cycle progression. Cells were treated for 48 h, and then the distribution of cells in the different phases of the cell cycle was determined by ﬂow cytometry. Sub-G1 represents apoptotic cells.*

P < 0.05 compared to the untreated group.# P < 0.05 compared to the genistein-treated group.

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Bax expression among all groups (data not shown). Thus, the ratio of anti-apoptotic/pro-apoptotic factor in the combination is the lowest of all groups.

3.6. Expression of angiogenic factors

Since human prostate cancer cells express a variety of angiogenic fac-tors, including VEGF165and VEGF121, which play important roles in new vessel formation, we examined whether the treatments used in this study affected the expression of these angiogenic factors by DU-145 cells. The cells were treated for not more than 36 h with genistein and/or terazosin, then angiogenic factor mRNA levels were measured by RT-PCR. The VEGF mRNA levels were extremely low in control and treated-groups at 6, 12, and 24 h. However, at 36 h treatment, terazosin caused a slight, but not signiﬁcant, decrease in VEGF165mRNA levels after calibration against b-actin mRNA, whereas both genistein and the combination caused a signif-icant decrease, the effect being greater with the combination. The same

Fig. 6. Expression of VEGF165and VEGF121in DU-145 cells. Treatment for 36 h, the expressions of VEGF isoforms were analyzed by RT-PCR. The VEGF mRNA expression was normalized to b-actin mRNA, then the density of the band was expressed as the relative density compared to that in untreated cells (control), taken as 100%.*

P < 0.05 compared to the genistein-treated group.

VEGF secretion (% of control) 0

20 40 60 80 100

*

#

*

T1 G5 T1+G5 C

Fig. 7. Effects of genistein and/or terazosin on VEGF secretion in DU-145 cells. The cells were treated for 36 h, and then VEGF secretion to medium was determined by ELISA method.*

P < 0.05 compared to the genistein-treated group. Fig. 4. Western blotting showing procaspase-3 activation and PARP

cleavage in DU-145 cells. After normalized to b-actin, the density of the band was expressed as the relative density compared to that in untreated cells (control), taken as 100%.*_{P < 0.05 compared to the untreated group.} #_{P < 0.05 compared to the genistein-treated group.}

Fig. 5. Western blotting of Bcl-XL expression in DU-145 cells. After normalized to b-actin, the density of the band was expressed as the relative density compared to that in untreated cells (control), taken as 100%.*

P < 0.05 compared to the genistein-treated group.

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trend was seen with VEGF121mRNA expression, but genistein alone had a greater effect on VEGF165levels than on VEGF121levels, whereas the com-bination did not (Fig. 6). Moreover, the protein levels of secreted VEGF in medium (Fig. 7) were in accordance with mRNA results.

4. Discussion

Prostate cancer is intrinsically heterogeneous and

con-sists of a simultaneous mixture of androgen-responsive

and androgen-unresponsive cells

[39,40]

. Soy products

containing isoﬂavones are widely available in food and

hu-mans consuming soy have micromolar concentrations of

isoﬂavones in the blood

[22,24,25]

. We recently showed

that soy isoﬂavones inhibit the growth of human bladder

and hepatoma cancers both in vitro and in vivo

[31–33]

.

In addition, soy genistein has been shown to inhibit the

growth of both benign and malignant prostate tissue

[35]

. On the other hand, terazosin has been proved to be

useful in the treatment of prostate cancer

[12–14,17]

. In

terms of induction of apoptosis or inhibition of angiogenic

factor expression, our results demonstrated that the

com-bination of genistein and terazosin was more effective than

either alone on hormone-independent human prostate

cancer cells. Although, the anticancer effects of terazosin

is gradually approved by many studies. But the reported

IC

50

of terazosin on prostate cancer cells is higher than

100 l

M (about 46

l

g/ml)

[12,13,19]

. We used lower and

nontoxic dosage of terazosin (1

l

g/ml) to decrease adverse

effects

[41–43]

, and in combination with 5

l

g/ml of

geni-stein to produce more effective anticancer results

suggest-ing that this combination strategy would be an attractive

clinical option.

Many anticancer agents and DNA-damaging agents

ar-rest the cell cycle at G1, S, or G2/M phase and induce

apop-totic cell death

[44–49]

. The cell cycle check-points

function to ensure that cells have time for DNA repair

[3,46,47]

, whereas apoptotic cell death functions to

elimi-nate irreparable or unrepaired damaged cells

[50]

. This

study showed that, as in other tumor cells

[30,33,34]

,

gen-istein arrested DU-145 cells in G2 phase. Of interest, this

G2 phase arrest by genistein was followed by increased

apoptotic death after co-treatment with terazosin. The

DU-145 cell line carries a mutation in p53 and the bax gene

[38]

. Studies have shown that alterations in the p53 gene

in tumor cells result in defective checkpoint function and

sensitize tumor cells to chemotherapy

[38,50]

. One

expla-nation for the increased sensitivity of p53-mutated tumor

cells is that the cells with DNA lesions prematurely enter

into mitosis because they are unable to regulate cyclin

B1/Cdc2 activity and cannot undergo G2 checkpoint arrest

[50]

. This study suggests that terazosin preferentially

over-rides the genistein-induced G2 checkpoint arrest in

DU-145 cells with defective p53 function. Moreover, our

unpublished data showed the increase in the other

check-point arrest gene p21 by genistein was reduced after

co-treatment with terazosin. On the other hand, the combined

effect of terazosin and genistein in reducing Bcl-X

L

levels

would explain, in part, why terazosin pushes the arrested

DU-145 cells towards apoptosis.

Human VEGF mRNA is transcribed from eight exons of a

single gene and is alternatively spliced into at least six

mRNAs, which give rise to mature proteins of 121, 145,

165, 183, 189, and 206 amino acids. 121 and

VEGF-165 are the best characterized and are the most abundant

in normal tissues, including blood vessels. Prostate tumors,

like most tumors, overexpress VEGF, thereby promoting

the development of tumor neovascularization

[51,52]

,

and this overexpression correlates with increasing grade,

vascularity, and tumorigenicity. Our results showed that

VEGF was highly expressed in DU-145 cells, as shown in

other studies

[52,53]

, and that genistein decreased VEGF

expression and that the combination was even more

effective.

Results of the present study clearly demonstrate that a

nontoxic dose of terazosin signiﬁcantly enhances the

anti-tumor activity of genistein on DU-145 human prostate

cancer cells suggesting that this combination could

poten-tially be useful in prostate cancer therapy. However, this

study is limited because it investigated only one prostate

cancer cell line, and the basis of the interaction of these

two compounds is not fully clear. It is not known whether

other in vivo experiments would have results comparable

to those determined in this culture study. More in vivo

studies are required to clarify whether the combined

treat-ment is an effective antitumor strategy.

Conﬂicts of interest statement

None declared.

Acknowledgements

This study was supported by the National Science

Coun-cil, Executive Yuan, Taiwan (Grant NSC

92-2314-B-242-006; NSC 93-2314-B-242-003 and NSC

93-2320-B-037-025; NSC 94-2320-B-242-011).

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