Phenethyl Isothiocyanate (PEITC) Promotes G2/M Phase Arrest via p53 Expression and Induces Apoptosis through Caspase- and Mitochondria-dependent Signaling Pathways in Human Prostate Cancer DU 145 Cells

Abstract.

Phenethyl isothiocyanate (PEITC), one of many

compounds found in cruciferous vegetables, has been

reported as a potential anticancer agent. In earlier studies,

PEITC was shown to inhibit cell growth and induction of

apoptosis in many cancer cell lines. However, no report has

shown whether PEITC can induce apoptosis in human

prostate cancer cells. Herein, we aimed to determine whether

PEITC has anticancer activity in DU 145 human prostate

cancer cells. As a result, we found that PEITC induced a

dose-dependent decrease in cell viability through induction

of cell apoptosis and cell cycle arrest in the G

/M phase of

DU 145 cells. PEITC induced morphological changes and

DNA damage in DU 145 cells. The induction of G

/M phase

arrest was mediated by the increase of p53 and WEE1 and it

reduced the level of CDC25C protein. The induction of

apoptosis was mediated by the activation of caspase-8-,

caspase-9- and caspase-3-depedent pathways. Results also

showed that PEITC caused mitochondrial dysfunction,

increasing the release of cytochrome c and Endo G from

mitochondria, and led cell apoptosis through a

mitochondria-dependent signaling pathway. This study showed that PEITC

might exhibit anticancer activity and become a potent agent

for human prostate cancer cells in the future.

Prostate cancer is not a single disease and is an umbrella

which under a plethora of heterogeneous diseases is

included. These heterogeneous diseases include indolent

localized tumors and aggressive metastatic diseases (1). In

the developed world, prostate cancer is the primary male

cancer, accounting for 24% of all diagnosed male cancer

cases, and is the second leading cause of death from cancer

(2). In Taiwan, prostate cancer is the seventh most common

cancer in males and about 8.0 men per 100 thousand die

annually from prostate cancer based on reports from the

Department of Health, Executive Yuan, R.O.C (Taiwan) in

Correspondence to: Jing-Gung Chung, Department of Biological

Science and Technology, China Medical University, No. 91, Hsueh-Shih Road, Taichung 404, Taiwan, R.O.C. Tel: +886 422053366 ext. 2161, Fax: +886 422053764, e-mail: [email protected]

Key Words: Phenethyl isothiocyanate, cell cycle arrest, apoptosis,

mitochondria, caspases, prostate cancer DU 145 cells.

ANTICANCER RESEARCH 31: 1691-1702 (2011)

Phenethyl Isothiocyanate (PEITC) Promotes G

₂

/M

Phase Arrest

via p53 Expression and Induces Apoptosis

through Caspase- and Mitochondria-dependent Signaling

Pathways in Human Prostate Cancer DU 145 Cells

NOU-YING TANG

, YA-TING HUANG

, CHUN-SHU YU

, YANG-CHING KO

,

SHIN-HWAR WU

, BIN-CHUAN JI

, JAI-SING YANG

, JIUN-LONG YANG

,

TE-CHUN HSIA

, YA-YIN CHEN

and JING-GUNG CHUNG

Schools of

Chinese Medicine and

Pharmacy, Departments of

Medical Laboratory Science and Biotechnology,

8

_{Pharmacology and}

_{Biological Science and Technology, Graduate Institutes of}

_{Clinical Medical Science,}

_{Chinese Pharmaceutical Science, China Medical University, Taichung 404, Taiwan, R.O.C.;}

4

_{Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine,}

St. Martin De Porres Hospital, Chiayi 600, Taiwan, R.O.C.;

5

_{Division of Critical Care Medicine, Department of Internal Medicine, and}

_{Division of Respiratory Care Center, Department of Medicine,}

Changhua Christian Hospital, Changhua 500, Taiwan, R.O.C.;

10

_{Department of Internal Medicine, China Medical University Hospital, Taichung 404, Taiwan, R.O.C.;}

_{Department of Chinese-Western Medicine Integration,}

Chung Shan Medical University Hospital, Taichung 402, Taiwan, R.O.C.;

12

_{School of Medicine, Chung Shan Medical University, Taichung 402, Taiwan, R.O.C.;}

_{Department of Biotechnology, Asia University, Taichung 413, Taiwan, R.O.C.}

2009. About 90% of advanced prostate cancer patients

developing skeletal metastases and in advanced prostate

cancer patients is morbidity from bone metastasis is high (3),

despite treatments including radiation, chemotherapy, or

combination of radiotherapy with chemotherapy.

The majority of cancer-targeting drugs cause aberrant

cellular growth through the induction of apoptosis (4-6).

Apoptosis, programmed cell death type I, is regulated by

multifactor and signaling pathways to remove unwanted

cells (7, 8). Active caspases are known to be biologically

important and associated with apoptosis (9).

Poly-ADP-ribose polymerase (PARP) is cleaved by both caspase-3 and

caspase-7, and is an abundant DNA-binding enzyme that

detects and signals DNA strand breaks (10). Caspase-3 in

particular is the main executor of apoptosis and caspase

activation is considered to be a key hallmark of apoptosis

(11). Cytochrome c release from the internal part of the

mitochondrial membrane into the cytosol leads to the

activation of caspase-9, -3, -6, and -7, and then to apoptosis

(12-14).

Traditional drugs obtained from various plant types with

active natural compounds such as licochalcone-A (15),

xanthoangelol (16) and chalcones (17) have been reported to

act as potential agents for the treatment of cancer. Dietary

intake of cruciferous vegetables can reduce the risk of various

types of malignancies (18). Phenethyl isothiocyanate (PEITC)

is one of such important compounds from cruciferous

vegetables. Several mechanisms have been shown to be

involved in the antitumor effect of PEITC, including the

inhibition of cytochrome p450 enzymes, the induction of

phase II detoxification enzymes (19) and the reduction of

azoxymethane-induced colonic aberrant crypt foci formation

(20). PEITC also inhibited

4-(methylnitrosamino)-1-(3-pyridyl)-1-butone-induced pulmonary neoplasia in A/J mouse

lung (21). In addition, PEITC can act as a cancer

chemopreventive in rat (22). Recently, it was reported that

PEITC inhibits oxidative phosphorylation to trigger reactive

oxygen species-mediated death of human prostate cancer cells

(23).

Although many reports stated that PEITC induced cell

death through induction of apoptosis, there is no report to

address whether this occurs in human prostate cancer cells.

In this study, we investigated the effects of PEITC on cell

growth of human prostate cancer cells.

Materials and Methods

Reagents. PEITC, RPMI-1640 medium, fetal bovine serum, and

penicillin and streptomycin antibiotic mixture were purchased from Invitrogen Life Technologies (Grand Island, NY, USA). Propidium iodide (PI), dimethyl sulfoxide (DMSO), RNase A, and 4,6-diamidino-2-phenylindole (DAPI) were from Sigma-Aldrich Corp. (St. Louis, MO, USA). The antibodies against BAK, BID, BCL-2, cell division cycle 25C (CDC25C), and apoptosis inducing effect

(AIF) were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), and the antibodies against cytochrome c, Glucose-regulated protein 78 (GRP78), and PARP were from BD PharMingen (Palo Alto, CA, USA). 6-Carboxy-2,7-dichlorodihydrofluorescein diacetate (H2DCFDA), Fluo-3/AM and 3,3’-dihexyloxacarbocyanine iodide (DiOC₆)were purchased from Invitrogen Life Technologies. The caspase-9 inhibitor (Z-LEHD-FMK), caspase-8 inhibitor (Z-IETD-FMK) and caspase-3 inhibitor (Z-DEVD-(Z-IETD-FMK) were obtained from R&D Systems Inc. (Minneapolis, MN, USA). All chemicals were reagent grade.

Cell culture. The human prostate carcinoma cell line (DU 145)

was purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan, ROC). DU 145 cells were maintained in RPMI-1640 medium with 2 mM L-glutamine supplemented with 10% heat-inactivated fetal bovine serum, 100 μg/ml penicillin and 100 U⁄ml streptomycin. Cells (1×105_{cells/ml) were}

maintained at 5% CO₂and 37˚C in humidified 5% CO₂and 95% air (24).

Cell morphology and cell viability assay. DU 145 cells were

maintained on 12-well plates and then incubation with different concentrations (0, 1, 5, 10, 15 and 20 μM) of PEITC for different time periods (24 and 48 h). For examination of cell morphological, treated and negative-control cells were observed at 24 h-incubation under a phase-contrast microscope and photomicrographs were taken. Cell viability was determined by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The DU 145 (1×104_{cells/well/100 μl) cells were cultured in 96-well plates. After}

incubation overnight, the RPMI-1640 medium in each well was replaced with different concentrated solutions of PEITC and cells further incubated for 24 and 48 h. At the end of incubation, 10 μl of MTT (5 mg/ml in phosphate-buffered saline (PBS)) (Sigma-Aldrich Corp.) were added individually to each well for 4 h incubation at 37˚C, and then removed media after 100 μl of DMSO were added. The absorbance was then measured by Microplate Reader (Bio-Rad, Hercules, CA, USA) at 570 nm (25, 26). In addition, cultured DU 145 cells with or without treatment with 25 μM of caspase-8 inhibitor (Z-IETD-FMK), caspase-9 inhibitor (Z-LEHD-FMK) or caspase-3 inhibitor (Z-DEVE-FMK), respectively, for 3 h prior to the addition 10 μM of PEITC were then analyzed for cell viability as described previously (26, 27).

Flow cytometric analysis for DNA content. DU 145 cells were

plated at a density of 2×105_{cells on 12-well plates for 24 h, and}

exposed to 0, 1, 5, 10, 15 and 20 μM of PEITC for different time periods. At the end of incubation, cells were trypsinized and centrifuged then the cell pellet from each treatment was resuspended with 1 ml of PI staining buffer containing 4 mM sodium citrate, 0.1% Triton X-100, 50 μg/ml PI and 200 μg/ml RNAse and incubated for 30 min at 37˚C in the dark. All samples were analyzed for cell cycle distribution and sub-G1 proportion (apoptosis) by using a FACSCalibur flow cytometer and BD CellQuest Acquisition software (San Jose, CA, USA) as described previously (24, 27). Data in each phase were reported as the percent age cells in each phase of the cell cycle.

DAPI staining. Morphological changes of apoptosis were examined by

staining DU 145 cell nuclei with DAPI. Cells (5×104_{cells) were}

of PEITC for 24 h. The cells were washed twice with PBS then were fixed with 4% paraformaldehyde-PBS solution for 15 min and stained with DAPI (300 nM) for 30 min at room temperature. The cells were washed with PBS and mounted. Images of DAPI fluorescence were examined and photographed under a fluorescence microscope as described previously (28, 29).

Comet assay. DU 145 cells (1×105_{cells) were treated with PEITC}

(0, 1, 5, 10, 15 and 20 μM) for 24 h and then cells were embedded onto 0.6% low melting point agar then immersed in ice-cold cell lysis solution (2.5 mM NaCl, 100 mM ethylenediaminetetra acetic acid (EDTA), 10 mM Tris (pH 10), 1% N-laurylsarcosine, 1% Triton X-100 and 10% DMSO). Cells were then washed and digested with Tang et al: Phenethyl Isothiocyanate Triggers Apoptosis in Human Prostate Cancer Cells

Figure 1. PEITC affected on cell morphology and viability in DU 145 cells. Cells were cultured in RPMI-1640 medium and 10% FBS with PEITC

for 24 and 48 h. Morphological changes were examined and cells photographed under a phase-contrast microscope (×200) (A). Cells viability was measured by flow cytometric assays as described in the Materials and Methods (B). *p<0.05 Significantly different from the control.

two units of endonuclease III in the same buffer for 1 h at 37˚C then slides were denatured with 0.3 mM NaOH, 1 mM EDTA (pH 13.4) for 20 min and electrophoresis was carried out at 25 V, 300 mA for 25 min. Cellular DNA was stained with 4 μg/ml PI and examined under a fluorescence microscope as described previously (26, 30).

Flow cytometric detection of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨ_m) and intracellular Ca2+

levels. DU 145 cells were plated at a density of 2×105_{cells on 12-well}

plates for 24 h, and then PEITC (10 μM) was added to the well and cells incubated for different time periods. At the end of incubation, cells were trypsinized and centrifuged. The cells from each treatment were collected and washed twice with PBS, re-suspended in 500 μl of H₂DCFDA (10 μM) for ROS, 500 μl of DiOC₆(1 μM) for ΔΨ_mand 500 μl of Fluo-3/AM (2.5 μg/ml) for intracellular Ca2+ _{release. All}

samples were incubated with these specific fluorochromes at 37˚C for

30 min and the differential expressions from each treatment were analyzed by flow cytometry (27, 31).

Caspase-3, -8 and -9 activity assays. DU 145 cells were plated at a

density of 2×105_{cells on 12-well plates for 24 h, and then PEITC (10}

μM) was added to the well and cells incubated for different time periods. The cells from each treatment were trypsinized and centrifuged, washed twice with PBS and were re-suspended in 50 μl of 10 μM substrate solution (PhiPhiLux-G₁D₁ for caspase-3, CaspaLux8-L₁D₂for caspase-8 and CaspaLux9-M₁D₂for caspase-9, OncoImmunin, Inc. Gaithersburg, MD, USA) before being incubated at 37˚C for 60 min. The cells were washed again by PBS and were analyzed by flow cytometry as described previously (29, 30).

Western blotting analysis. Western blotting was carried out as

previously described (30). DU 145 cells were seeded at a density of 2×106_{cells/ml in RPMI-1640 medium with 10% fetal bovine serum}

for 24 h. Cells were treated with or without 10 μM PEITC for 0, 6, 12, 24 and 48 h. Cells from each treatment were collected and protein was extracted into the PRO-PREP™ protein extraction solution (iNtRON Biotechnology, Seongnam, Gyeonggi-Do, Korea), and protein concentrations were determined by a BCA Protein Assay kit (Bio-Rad Laboratories, Hercules, CA, USA) (26). Proteins were resolved on a polyacrylamide gel via electrophoresis (SDS-PAGE). After SDS-PAGE transfer, the Polyvinylidene Fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA, USA) was stained by primary antibody (anti-CDC25C, WEE1, p53, FAS, Fad ligand, BCL-2, BAK, BID, PARP, cytochrome c, AIF, GRP78 and GADD153), washed and then stained by appropriate horseradish peroxidase–conjugated secondary antibodies (GE Healthcare Pewaukee, WI, USA) for enhanced chemiluminescence (ECL) reagent (Millipore) as described previously (29, 32).

Immunofluorescence staining and confocal laser scanning microscopy. DU 145 cells (5×104 _{cells/well) were placed on 4-well}

chamber slides before being treated with 10 μM PEITC for 24 h. Cells were then fixed in 3% formaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 1 h with blocking of non-specific binding sites using 2% bovine serum albumin (BSA) as described previously (29, 33). Fixed cells were stained with primary antibodies to GADD153 and endonuclease G (Endo G) (1:200 dilution) overnight and then stained with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG secondary antibody at 1:100 dilution) (green fluorescence) followed by mitochondria and nuclei as counterstaining with PI (red fluorescence). Photomicrographs were obtained using a Leica TCS SP2 confocal spectral microscope.

Statistical analysis. The data are presented as the mean±S.D. of three

independent experiments. The statistical significance of the mean difference between the control and treated groups was determined by a paired t-test. P<0.05 was considered statistically significant.

Results

PEITC induced morphological changes and reduced the

percentage of viable DU 145 cells. To evaluate the effect of

PEITC on DU 145 prostate cancer cells, we determined and

measured the cell morphological changes and viability using a

phase-contrast microscope and the MTT assay, respectively. The

DU 145 cells. Cells were placed in RPMI-1640 and 10% FBS with PEITC for 48 h then were harvested for analysis of cell cycle distribution (A) by flow cytometric assay. Cells were harvested for examination of the G₂/M phase arrest-associated protein levels and were assayed by Western blotting (B) as described in the Materials and Methods.

cells were dose-dependently treated with PEITC and

morphological changes of DU 145 cells after treatment with

PEITC are shown in Figure 1A. Compared with the control

(untreated) cells, the majority of the PEITC-treated DU 145 cells

became irregularly shaped spinouts and less cell number (Figure

1A). These morphological changes demonstrated cell damage

after PEITC treatment. As shown in Figure 1B, PEITC

significantly reduced the viability of DU 145 cells in a time- and

dose-dependent manner. A sharp decrease in cell viability was

present at 5 μM of PEITC treatment time. The cytotoxic effect

was more evident at 48 h. The viability of DU 145 cells after

exposure to 10 μM of PEITC for 48 h decreased by almost 50%.

PEITC induced cell cycle arrest and apoptosis in DU 145

cells. The effects of PEITC on the cell cycle progression in

DU 145 cells were determined by flow cytometry and PI

staining. In DU 145 cells, treatment with PEITC for 48 h

resulted in a dose-dependent increase in the percentage of

Figure 3. PEITC induced apoptosis and DNA damage in DU 145 cells. Cells were treated with PEITC at various doses and then were incubated for

24 h. Cells were harvested for DAPI staining then were examined and photographed under a fluorescence microscope for examination the apoptosis (A). Cells were stained with PI to examine DNA damage by Comet assay (B) as described in the Materials and methods.

Figure 4. PEITC affected the levels of mitochondria membrane potential (ΔΨm), production of reactive oxygen species (ROS) and Ca2+and in DU

145 cells. Cells were treated with 10 μM of PEITC for various time periods then were collected and stained by DiOC6 for the ΔΨmlevels (A), with

2,7-dichlorodihydrofluorescein diacetate for ROS production (B) and by Fluo-3 for Ca2+_{level (C) as described in the Materials and Methods. Data}

Tang et al: Phenethyl Isothiocyanate Triggers Apoptosis in Human Prostate Cancer Cells

Figure 5. PEITC induced caspase-3, -8 and -9 activities in DU 145

cells. Cells were treated with 10 μM of PEITC for 12, 24, 48 or 72 h and cells were collected for determination activities of caspase-3 (A), caspase-8 (B) and caspase-9 (C) as described in the Materials and Methods. Data represents the mean±S.D. of three experiments. *p<0.05, significantly different from the control.

Figure 6. Caspase inhibitors protected against PEITC-induced

cytotoxicy in DU 145 cells. Cells were pretreated with the caspase-9 LEHD-FMK) (A), caspase-8 IETD-FMK) (B) and caspase-3 (Z-DEVE-FMK) (C) inhibitors, and then were treated with 10 μM PEITC for 24 h before determination of viable cells as described in the Materials and Methods. Data represent the mean±S.D. of three experiments. *p<0.05, significantly different from the control.

cells in the G

/M phase and a concomitant reduction of cell

numbers in the S phase (Figure 2A). Higher concentrations

of PEITC (15 and 20 μM) retuned the proportion of G

/M

cells back to control levels. However, the proportion of G

/G

phase cells increased at treatment of PEITC at 15 and 20 μM.

The sub-G

phase was clearly to increase when the treatment

of PEITC was increased and in a dose-dependent manner

(Figure 2A). This was further verified by microscopic

examination that showed a decrease of viable cell number in

treated samples. Results from Western blotting shown in

Figure 2B also show that PEITC increased the protein level of

p53, but it inhibited the protein levels of CDC25C and WEE1.

Taken together, these data indicated that PEITC induced

apoptosis and arrested cell cycle progression at the G

/M

stage in DU 145 cells.

PEITC induced apoptosis and DNA damage in DU 145 cells.

We found that PEITC induced apoptosis in a dose-dependent

manner in DU 145 cells (Figure 3A). PEITC-induced DNA

damage was examined by Comet assay in DU 145 cells

(Figure 3B). Using this assay, we found that PEITC-induced

DNA damage in a dose-dependent manner in DU 145 cells.

PEITC reduced the level of ΔΨ

and increased the production

of ROS and Ca

in DU 145 cells. To investigate whether

PEITC induced cytotoxic effects involved in ΔΨ

, ROS and

Ca

in DU 145 cells, the levels of ΔΨ

and ROS and Ca

were measured by flow cytometry and the results are shown in

Figure 4. The data indicate that PEITC significantly reduced

the level of ΔΨ

within 48 and 72 h treatment of PEITC

(Figure 4A) and promoted ROS production at 6 and 12 h

treatment of PEITC (Figure 4B). However, treatments for 24 h

led to a decrease the ROS levels when compared to the control

group, but not significantly so. Furthermore, PEITC increased

the intracellular Ca

level at 0.5-6 h treatment (Figure 4C) in

DU 145 cells.

PEITC promoted the activities of caspase-3, -8 and -9 in DU

145 cells. To determine whether PEITC-induced apoptosis

was associated with caspase activation in DU 145 cells, the

activities of caspase-3, -8 and -9 in DU 145 cells after

exposure to PEITC were assayed by flow cytometry and the

results are shown in Figure 5. The results indicate that

PEITC promoted the activities of caspase-3 at 24-72 h in a

time-dependent manner (Figure 5A), increased the caspase-8

PEITC. Cells were treated with 10 μM of PEITC for 0, 6, 12, 24 and 48 h before the total proteins were determined, as described in Materials and Methods. The levels of associated protein expressions (A: FAS ligand and FAS; B: BCL-2, BAK and BID; C: PARP, AIF and cytochrome c; D: GRP78 and GADD153) were estimated by Western blotting, as described in the Materials and Methods.

activity at 48 h, (Figure 5B), and that of caspase-9 at 24 h

(Figure 5C). Based on these observations, we suggest that

caspases might be involved in the induction of apoptosis in

DU 145 cells after exposure to PEITC.

Caspase-specific inhibitors protected against PEITC-induced

cell death in DU 145 cells. DU 145 cells were pretreated with

the caspase-3 inhibitor DEVE-FMK), caspase-8 inhibitor

(Z-IETD-FMK) and caspase-9 inhibitor (Z-LEHD-FMK),

Figure 8. PEITC altered the GADD153 and Endo-G distributions in DU 145 cells. Cells were incubated with 10 μM of PEITC for 24 h, and then

were fixed and stained with primary antibodies to GADD153 (A) and Endo G (B) which were stained by FITC-labeled secondary antibodies (green fluorescence) and the proteins were detected by a confocal laser microscopic system. The nuclei were stained by PI (red fluorescence). Areas of co-localization of GADD153 and Endo G expressions in the merged panels are yellow. Scale bar, 20 μm.

respectively, and then cells were harvested for measuring the

percentage of viability and the results are shown in Figure 6.

These data indicate that caspase-8 inhibitor (Z-IETD-FMK)

significantly increased the percentage of viable cells (Figure 6B),

while the other two inhibitors did not (Figure 6A and C) after

exposure to PEITC.

PEITC affected the apoptosis-associated protein levels in DU

145 cells. The effects of PEITC on the apoptotic associated

protein levels in DU 145 cells were assayed by Western

blotting and the results are shown in Figure 7. The results

indicate that PEITC increased the protein levels of FAS

ligand, FAS (Figure 7A), BAK (Figure 7B), AIF, cytochrome

c (Figure 7C) and GRP78 (Figure 7D), but reduced the levels

of BCL-2 and BID (Figure 7B), PARP (Figure 7C) and did

not affect GADD153 (Figure 7D) level in DU 145 cells.

PEITC altered the distribution of GADD153 and Endo G in

DU 145 cells. The effects of PEITC on the levels and

distribution of apoptosis-associated proteins GADD153 and

Endo G in DU 145 cells were examined by confocal laser

microscope and the results are shown in Figure 8. PEITC

increased the protein levels of GADD153 (Figure 8A) and

Endo G (Figure 8B) and increased their translocation to nuclei.

Discussion

PEITC has been isolated from various common

vegetables, especially cruciferous, and has anticancer

activities against many human cancer cell types in vitro

and in vivo (34-39). In this study, we found that PEITC

exhibited antitumor activity as it significantly inhibited

prostate cancer cell growth and induced apoptosis, and

more importantly, through mitochondrialdependent and

-independent pathways.

It is well known that cell proliferation and viability are

one of major observations for the assessment of cytotoxicity

and screening of anticancer drugs. Our results also show that

PEITC induced morphological changes and reduced the

percentage of viable of DU 145 cells (Figure 1A and B).

Apoptosis is one form of programmed cell death and is

characterized by morphological changes, chromatin

condensation and extensive DNA fragmentation. The

frequency and time of appearance of apoptosis depend on the

apoptosis-inducing signal and cell types (40, 41). It was

reported that failure of apoptosis in cancer cells may promote

survival and accumulation of cells to form tumors (42).

Therefore, the induction of apoptosis in cancer cells has

become a strategy for their elimination (43, 44).

In the present study, we investigated the effect of PEITC

on apoptosis in DU 145 human prostate cancer cells in vitro.

Flow cytometric assay for sub-G

phase examination, DAPI

staining and the comet assay were used to study the

morphological changes of apoptosis and DNA damage,

respectively. Results showed that PEITC induced sub-G

phase in DU 145 cells and the data from DAPI staining also

confirmed that PEITC induced DU 145 cells apoptosis and

that the DNA damage occurred in the cell apoptotic process.

To further verify the DNA damage induced by PEITC, the

single-cell gel electrophoresis assay (Comet assay) was

performed and the results showed that PEITC induced DNA

damages dose dependently. We suggest that DNA damage

might be one of the means by with apoptosis is induced by

PEITC in DU 145 cells.

It is also reported that ROS are involved in apoptosis in

cancer cells (45). Thus, we used flow cytometric assay and

found that PEITC promoted the release of ROS in DU 145

cells (Figure 4B) at 6-12 h treatment. The results also

showed that PEITC reduced the levels of ΔΨ

and

resulted in Ca

increased in DU 145 cells (Figure 4A and

C). These observations indicate that PEITC-induced

apoptosis also involves mitochondria. It is well known that

caspases also play important roles in apoptosis, and we

investigated the activities of caspases and found that

PEITC significantly activated of caspase-3, -8 and -9

(Figure 6A, B and C). PEITC-induced apoptosis involves a

caspase-dependent pathway. Caspase-3 is a critical enzyme

in the execution phase of apoptosis and a hallmark of

apoptosis (46). Our findings are in agreement with this

showing a significant increase in caspase-3 activity in DU

145 cells treated with 10 μM PEITC for 24 h, but no

change in caspase-8 protein expression (Figure 5B). This

shows that the treatment time was insufficient for an

increase in protein expression but sufficient for

enhancement of activity.

The BCL-2 family of proteins are the most important

regulators of apoptosis. BAX is an apoptotic agonist that

promotes cells’ apoptosis. BCL-2 is an intracellular

suppressor of apoptosis, which prolongs cell survival.

Therefore, the ratio of BCL-2/BAX of the cells will

determine whether or not apoptosis occurs (47).

In conclusion, our results clearly indicate that PEITC

promoted the expression of BAX but inhibited the expression

of BCL-2 in DU 145 cells thus contributing to the activation

of caspase-3 and the induction of apoptosis via the

mitochondrial apoptosis pathway (Figure 9).

Acknowledgements

This study was supported in part by Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH100-TD-B-111-004).

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Received January 29, 2011

Revised April 6, 2011

Accepted April 7, 2011